Biosensor for detecting smell, scent, and taste

ABSTRACT

The invention relates to biosensors for detecting odorants, especially a biosensor that mimics odorant detection by a mammal, for example, humans, dogs or cats. The field of the invention also related to the standardization of odors for scent, smell and taste using the biosensor of the invention, and the discovery of agonists, antagonists, and mixtures of odorants for creating new odors, masking odors, enhancing odors, and designing odors.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application Ser.No. 62/299,005, filed on Feb. 24, 2016, the disclosure of which ishereby incorporated by reference in its entirety.

REFERENCE TO SEQUENCE LISTING, TABLE OR COMPUTER PROGRAM

The official copy of the Sequence Listing is submitted concurrently withthe specification as an ASCII formatted text file via EFS-Web, with afile name of “ARX007_ST25.txt”, a creation date of Feb. 23, 2017, and asize of 45 kilobytes. The Sequence Listing filed via EFS-Web is part ofthe specification and is incorporated in its entirety by referenceherein.

BACKGROUND OF THE INVENTION

The olfactory receptor genes have been characterized through homology asseven transmembrane domain G protein-coupled receptors (GPCR). It isestimated that there are probably 500-750 olfactory receptor genesequences in humans, while there are 500-1000 olfactory genes in rat andmouse. Olfactory receptors are concentrated on the surface of the mucuscoated cilia and odorant molecules bind to the olfactory receptors inthe olfactory epithelium. Since mammals can detect at least 10,000 odorsand there are approximately 1,000 or fewer olfactory receptors, manyodorants must interact with multiple olfactory receptors.

The discriminatory power of olfactory receptors is such that it canperceive thousands of volatile chemicals as having different odors. Itis known that the olfactory system uses a combinatorial receptor codingscheme to decipher the odor molecules. One olfactory receptor canrecognize multiple odorants and one odorant is recognized by multipleolfactory receptors. A slight structural change in the odorant or achange in the concentration of the odorant in the environment results ina change in the odor-code of these receptors.

Each mammalian olfactory receptor neuron encodes only one olfactoryreceptor. The axons of the neurons expressing the same olfactoryreceptor converge to one olfactory bulb, which then processes theinformation to the brain. Olfactory receptors are structurally similarto G-Protein Coupled Receptors (GPCRs) and contain seven transmembrane(TM) domains connected by loops. The functionally important residues arepresent on the transmembrane helices 2-7.

Odor molecules belong to a variety of chemical classes: from alcohols,aldehydes, ketones and carboxylic acids to sulphur-containing compoundsand essential oils. The physicochemical descriptors of odor moleculesplay an important role in the prediction of odor response by theolfactory receptor. Very identical olfactory receptor sequences can havea structural bias for ligand specificity on the basis of the number ofcarbon atoms present in the ligands. About 8000 odorants have beenidentified in food. About 400 food odorants have been characterized andthis number approximately equals the number of olfactory receptors foundin humans. The response of mixtures of odorants is neither the additivenor an average of its components. Some mixtures lead to the emergence ofnovel perceptual qualities that were not present in the individualcomponents.

It is an object of this invention to provide a biosensor that producesan aromagraph for a mixture, composition, or molecule. It is an objectof the invention to provide a biosensor that is used to deconstruct thecomponents of a mixture, composition or molecule that are responsiblefor the scent, smell, odor, aroma, and/or taste of the mixture,composition or molecule. It is also an object of the invention toprovide a biosensor that is used to diagnose and remediate malodors.

SUMMARY OF THE INVENTION

The invention relates to biosensors for the detection of interactions atan Olfactory Receptor. In some embodiments, the invention relates to theuse of the biosensors of the invention to detect the interaction ofodorants at Olfactory Receptors. In some embodiments, a plurality ofbiosensors are used to detect the interaction of an odorant at aplurality of Olfactory Receptors. In some embodiments, the plurality ofbiosensors represent the repertoire or a portion of the repertoire of ananimals Olfactory Receptors. In some embodiments, the plurality ofbiosensors represent the repertoire or a portion of the repertoire ofhuman Olfactory Receptors. In some embodiments, the plurality ofbiosensors represent the portion of the repertoire of human OlfactoryReceptors that detect odorants in solution. In some embodiments, theplurality of biosensors represent the portion of the repertoire of humanOlfactory Receptors that detect odorants in the gaseous phase. In someembodiments, the plurality of biosensors represent the portion of therepertoire of human Olfactory Receptors that detect a particularodorant. In some embodiments, the plurality of biosensors represent theportion of the repertoire of human Olfactory Receptors that detect aparticular class or type of odorant.

In some embodiments, individual biosensors are comprised of an OlfactoryReceptor that is fused in its N-terminal region to a polypeptidesequence that targets the nascent polypeptide to the host cell membrane,and fused in its C-terminal region to a polypeptide that stabilizes thereceptor. In some embodiments, the polypeptide fused to the C-terminalregion of the Olfactory Receptor targets the receptor to theoutermembrane of the host cell. In some embodiments, the N-terminalportion of the biosensor is from positions 1-55 of the rat olfactoryreceptor RI7 (RI7). In some embodiments, full length olfactory receptorsare used without fusing to another protein. In some embodiments, thenucleic acids encoding the RI7 portion of the fusion receptor or thefull length olfactory receptor are preceded by a nucleic acid encodingan eight amino acid FLAG tag (Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys (SEQ IDNO:1)), e.g., used for identification and purification of the olfactoryreceptor fusion protein. In some embodiments, the Olfactory Receptorsequences are followed by a cassette encoding Green Fluorescent Proteinor Red Fluorescent protein. The GFP or RFP is fused to the olfactoryreceptor to allow detection of the location of the olfactory receptorwhen it is expressed in host cells. In some embodiments, the OlfactoryReceptor is a mammalian olfactory receptor. In some embodiments, theOlfactory Receptor is a human Olfactory Receptor. In some embodiments,the human olfactory receptor is OR1A1, OR2W1, OR2J2, OR5P3, or OR6A2. Insome embodiments, the ORF sequence fused in the construct comprisesnucleic acids encoding the amino acids sequence of the OlfactoryReceptor from amino acid position 56 to the end of the amino acidsequence.

In some embodiments, a construct is used for accepting ORF cassettes tomake new biosensors of the invention. In some embodiments the ORFcassette is fused into the construct so full length human receptorprotein is encoded in the construct. In some embodiments, the nucleicacid encoding the full length human olfactory receptor is fused in framewith a nucleic acid encoding an eight amino acid FLAG tag,(Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys (SEQ ID NO:1)). In some embodiments,the Olfactory Receptor sequences are followed by a cassette encodingGreen Fluorescent Protein or Red Fluorescent protein.

In some embodiments, the biosensors of the invention are also comprisedof a G-protein and an adenylate cyclase. In some embodiments, theG-protein is comprised of three subunits the Gα subunit, Gβ subunit, andGγ subunit. In some embodiments, the adenylate cyclase and the G proteinare from the same species. In some embodiments, the adenylate cyclaseand the G protein are from different species. In some embodiments, the Gprotein subunits are from the same or from different species. In someembodiments, the Olfactory Receptor, G protein and adenylate cyclase arefrom the same species, and in some embodiments, one or more of thecomponents are from different species. In some embodiments, thebiosensor polypeptides of the invention include polypeptides that have70%, 80%, 90%, 95%, and 99% sequence homology with SEQ ID NO: 6-10 and12-17.

In some embodiments, the biosensor of the invention includes apolypeptide that directly or indirectly produces a reporter molecule. Insome embodiments, the biosensor of the invention includes a polypeptidethat is the reporter molecule (e.g., an optical reporter). In someembodiments, the polypeptide is an adenylate cyclase. In someembodiments, the reporter molecule is cAMP. In some embodiments, thereporter(s) produced by the biosensor has a dynamic range of six toseven orders of magnitude, and the biosensor coupled to the reporter candetect binding of odorants and other molecules in a range of 0.15 partsper billion to about 420,000 parts per billion, or 10⁻⁹ M to about 10⁻³M. In some embodiments, the window of detection is six to seven ordersof magnitude within the range of 10 M to 10⁻¹² M. In some embodiments,the biosensor of the invention has a dynamic range of nine to ten ordersof magnitude. In some embodiments, the biosensor of the invention candetect binding of odorants and other molecules in a range of 10⁻¹¹ M toabout 10⁻² M. In some embodiments, the window of detection is nine toten orders of magnitude within the range of 10 M to 10⁻¹² M. In someembodiments, the biosensor of the invention has a dynamic range of threeto five orders of magnitude. In some embodiments, the window ofdetection is three to five orders of magnitude within the range of 10 Mto 10⁻¹² M. In some embodiments, the biosensor of the invention has adynamic range of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 orders of magnitude.

The invention also relates to nucleic acids encoding the biosensors ofthe invention. In an embodiment, the nucleic acids of the inventioninclude nucleic acids that hybridize under stringent hybridizationconditions to nucleic acids encoding one of the biosensor polypeptidesof the invention. In an embodiment, the biosensor polypeptides of theinvention include the polypeptides encoded by nucleic acids thathybridize under stringent hybridization conditions to nucleic acidsencoding the biosensor polypeptides describe above. In an embodiment,the nucleic acids of the invention encode a polypeptide of one of SEQ IDNOS: 6-10 and 12-17, or are a nucleic acid that hybridizes understringent hybridization conditions to a nucleic acid encoding apolypeptide of one of SEQ ID NOS: 6-10 and 12-17. In an embodiment, thenucleic acids of the invention encode a polypeptide that has 70%, 80%,90%, 95%, and 99% sequence identity with one of SEQ ID NOS: 6-10 and12-17.

The invention relates to the biosensor polypeptides and biosensornucleic acids contained within host cells. In some embodiments, the hostcells are eukaryotic cells. In some embodiments, the host cell is afungal cell, animal cell, plant cell, or algae cell. In someembodiments, the fungal cell is selected from Saccharomyces, Pichia,Aspergillus, Chrysosporium, or Trichoderma. In some embodiments, thefungal cell is Saccharomyces cerevisiae, Pichia pastoris, Aspergillusniger, Aspergillus oryzae, Chrysosporium lucknowense, or Trichodermareesei. In some embodiments, the host cell is a mammalian cell linederived from Chinese hamster cells, Human kidney cells, Monkey kidneycells, Human cervical cancer cells, or Mouse myeloma cells. In someembodiments the host cell is a human cell. In some embodiments, the hostcell is a murine cell. In some embodiments, the host cell is a caninecell.

The invention also relates to the use of host cells containing thebiosensor polypeptide and biosensor nucleic acids of the invention. Insome embodiments, the invention relates to the use of membrane fractionswith the biosensor made from host cells with the biosensor. In someembodiments, a reference receptor-reporter is included in the host cellor membrane fraction to allow relative, real-time measurements to bemade on the biosensor of the invention. In some embodiments, real timemeasurements are used to measure the interaction of an odorant with atleast one Olfactory Receptor. In some embodiments, real timemeasurements are used to measure the interaction of an odorant at aplurality of different Olfactory Receptors. In some embodiments, realtime measurements are used to measure the interaction of a plurality ofdifferent odorants at the same or different Olfactory Receptors. In someembodiments, real time measurements are made and compared to a referenceto provide comparative numbers for the interaction of an odorant atOlfactory Receptors. In some embodiments, real time measurements aremade and compared to a reference to provide comparative numbers for theinteraction of different odorants at the same or different OlfactoryReceptors. In some embodiments, quantitating versus a reference willproduce aromagraphs for molecules, mixtures, and/or compositions thatcan be compared and contrasted. In some embodiments, the reportermonitored in real time is an optical reporter. In some embodiments, thereporter monitored in real time is a nonoptical reporter. In someembodiments, the reference is a G-protein coupled receptor with a knownaffinity for a known ligand. In some embodiments, the reference receptorhas an activity range of 1, 2, 3, 4, 5, 6, 7, 8, or 9 orders ofmagnitude. In some embodiments, the reference receptor has an activityrange of 2-4 orders of magnitude and 4-5 different reference receptorsare used to cover the activity range of Olfactory Receptors from 10 M to10⁻¹² M. In some embodiments, the reference receptor-ligand pair isselected to have an affinity that is similar to the affinity of thetested Olfactory Receptor-odorant pair. In some embodiments, theconcentration of the tested odorant is varied in a series of wells overa range of 3, 4, 5, 6, 7, 8, 9, or 10 orders of magnitude and arecompared to a reference. In some embodiments, the concentration of thetested odorant is varied in a series of wells over a range of 3 to 10orders of magnitude and are compared to a reference. In someembodiments, the Olfactory Receptors are identified as having strong,intermediate, or weak interactions with the tested odorant. In someembodiments, the reference receptor is associated with a reporter thatis different from the reporter associated with the OlfactoryReceptor(s). In some embodiments, the different reporters are opticalreporters. In some embodiments, the different optical reporters aremonitored in real time in the same reaction.

In some embodiments, the biosensors of the invention are used toidentify odorants in a mixture or composition that contribute to thescent, odor, smell, aroma, or taste of the mixture or composition. Insome embodiments, the biosensor of the invention is used to characterizethe scent, odor, smell, aroma, or taste of a composition, mixture, or aplurality of mixtures and/or compositions. In some embodiments, thebiosensors are used to make a formulation or recipe for a composition ormixture that has a characteristic scent, odor, smell, aroma, or taste.In this embodiment, a component of a composition or a mixture may bereplaced by a different component(s) without a loss of thecharacteristic scent, odor, smell, aroma, or taste by using thebiosensor to find replacement component(s) that have similarinteractions at the Olfactory Receptors as the replaced component. Insome embodiments, the replacement component is from a natural source(replacing a non-natural component(s)). In some embodiments, thereplacement component is considered to be healthy and replaces acomponent considered to be unhealthy. In some embodiments, thereplacement component costs less. In some embodiments, the replacementcomponent is easier to manufacture or has better properties. In someembodiments, the replacement component substitutes for an ingredientthat has been banned or otherwise becomes expensive, unavailable, orhard to acquire. In some embodiments, the replacement componentsubstitutes for two or more ingredients in the composition. In someembodiments, the composition is completely reverse engineered and a setof components that produce the composition and its characteristic scent,smell, odor, aroma, and/or taste is designed.

In some embodiments, the biosensors of the invention are used to makelibraries of molecules with known aromagraphs that can be used to buildor maintain designer or desired scents, odors, smells, aromas, ortastes. In some embodiments, molecules from a library with or withoutknown aromagraphs can be used to build mixtures or compositions thatmimic the aromagraphs of desired known mixtures or compositions. In someembodiments, molecules from a library can be used to build mixtures orcompositions that meet an aromagraph specification. In some embodiments,the aromagraph is for a known mixture, composition or molecule. In someembodiments, the aromagraph is for a designed or theoretical scent,smell, odor, aroma, and/or taste. In some embodiments, the aromagraph isfor a desired modification of a known mixture, composition, or molecule.In some embodiments, the molecules from the library can also be used toremediate malodors.

In some embodiments, the biosensors of the invention are used tostandardize scents, smells, odors, aromas, and/or taste. For example,aromagraphs can be used to quantify and characterize different scents,smells, odors, aromas, and/or tastes creating a common language anddescription for the comparing and contrasting compositions, mixtures,and/or molecules. In some embodiments, the biosensors of the inventionare used to quantify the interaction of a scent, smell, odor, aroma,and/or taste with the OR repertoire. These quantized interactions can beused to describe a scent, smell, odor, aroma, and/or taste. For example,hundreds of vanilla flavoring or vanilla extract products advertise thatthey provide vanilla flavor (or smell, scent, aroma). The biosensors ofthe invention will quantify the interaction of these vanilla productswith the OR repertoire and permit standardization of these products on afunctional basis. A core set of OR interactions will define the vanillaresponse, with many minor or side OR interactions producing thedifferences between these vanilla products. Standardization also allowsthe quantitative description of scent, smell, aroma, odor, and/or tastefor purposes of branding, trademarks, and/or copyrights. Standardizationalso allows the quantitative description of new scents, smells, aromas,odors, and/or tastes.

In some embodiments, the biosensors of the invention are used toremediate a malodor. In this embodiment, the biosensor can be used tofind a molecule(s) that can mask a malodor (e.g., from sports equipment,clothing or shoes, or products that depend on scent/smell). In someembodiments, the masking molecule binds to but does not activate (in thesame way) the Olfactory Receptors which are causing the malodor scent.In this embodiment, the masking molecule can block the OlfactoryReceptor(s) that perceive the malodor. In some embodiments, the maskingmolecule activates other Olfactory Receptors that in combination withthe Olfactory Receptors activated by the malodor changes the perceptionof the malodor to a positive or null perception.

In some embodiments, the biosensors of the invention are used to createa composition or mixture with a desired scent, smell, odor, aroma,and/or taste. In some embodiments, the biosensors of the invention areused to make a composition or mixture that suppresses appetite. In thisembodiment, the mixture or composition that suppresses appetite isplaced in a device that creates an aerosol for delivering the appetitesuppressant to the subject. In some embodiments, the biosensors of theinvention are used to adjust the scent, smell, odor, aroma, and/or tasteof a product to suit the palates of subjects in different geographiclocations and/or different demographic groups. In some embodiments, adesired scent, smell, odor, and/or aroma is released from solid thatcontains the components for the desired scent, smell, odor, and/oraroma. In some embodiments, air freshener delivery systems are used toprovide the desired scent, smell, odor, and/or aroma (e.g., gladeplug-ins or other air freshener products).

In some embodiments, the biosensors of the invention are used tocharacterize aromascapes or odorscapes by identifying the components ofan aromascape or odorscape that cause a desired perception by somesubjects. In some embodiments, the biosensors of the invention are usedto build aromascapes or odorscapes that produce a desired response fromcertain subjects. In some embodiments, certain subjects will find thearomascape or odorscape appealing, repellent, arousing, relaxing, orother desired state for producing a desired behavior. In someembodiments, the aromascape or odorscape is used to brand a product orservice.

In some embodiments, the quantified scent, smell, odor, aroma, and/ortaste is used to identify the scent, smell, odor, aroma, and/or taste.In some embodiments, the quantified scent, smell, odor, aroma, and/ortaste is used to uniquely identify the scent, smell, odor, aroma, and/ortaste. In some embodiments, the quantified scent, smell, odor, aroma,and/or taste is used to communicate about the scent, smell, odor, aroma,and/or taste. In some embodiments, the quantified scent, smell, odor,aroma, and/or taste is used in a transaction for the scent, smell, odor,aroma, and/or taste. In some embodiments, quantified scent, smell, odor,aroma, and/or taste is used to describe a product or service. In someembodiments, quantified scent, smell, odor, aroma, and/or taste is usedto market a product or service. In some embodiments, quantified scent,smell, odor, aroma, and/or taste is used to sell a product or service.In some embodiments, quantified scent, smell, odor, aroma, and/or tasteis used to buy a product or service.

In some embodiments, an aromagraph is used to identify the scent, smell,odor, aroma, and/or taste. In some embodiments, an aromagraph is used touniquely identify the scent, smell, odor, aroma, and/or taste. In someembodiments, an aromagraph is used to communicate about the scent,smell, odor, aroma, and/or taste. In some embodiments, an aromagraph isused in a transaction for the scent, smell, odor, aroma, and/or taste.In some embodiments, an aromagraph is used to describe a product orservice. In some embodiments, an aromagraph is used to market a productor service. In some embodiments, an aromagraph is used to sell a productor service. In some embodiments, an aromagraph is used to buy a productor service.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts a plasmid map for some OR constructs.

DETAILED DESCRIPTION OF THE INVENTION

Before the various embodiments are described, it is to be understoodthat the teachings of this disclosure are not limited to the particularembodiments described, and as such can, of course, vary. It is also tobe understood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting, since the scope of the present teachings will be limited onlyby the appended claims.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present teachings, some exemplarymethods and materials are now described.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which can be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentteachings. Any recited method can be carried out in the order of eventsrecited or in any other order which is logically possible.

Definitions

As used herein, an “aromagraph” refers to a digital representation ofthe response to an odorant by a repertoire of Olfactory Receptors.

As used herein, “aromascape” and “odorscape” are used interchangeablyand both refer to the odors, aromas, smells and/or scents in theenvironment of a location. An aromascape or odorscape can be naturallyoccurring or engineered to produce a desired response(s) from anindividual or group of individuals. Aromascapes or odorscapes can beareas of a place of business such as a store, hotel, or sports arena,outdoor areas such as those found in parks, sports or entertainmentstadiums,

As used herein, an “effective amount” refers to an amount of a compound,formulation, material, or composition, as described herein effective toachieve a particular biological result.

As used herein, the terms “express” or “expression” refer to theproduction of a protein product from the genetic information containedwithin a nucleic acid sequence.

As used herein, an “expression vector” and an “expression construct” areused interchangeably, and are both defined to be a plasmid, virus, orother nucleic acid designed for protein expression in a cell. The vectoror construct is used to introduce a gene into a host cell whereby thevector will interact with polymerases in the cell to express the proteinencoded in the vector/construct. The expression vector and/or expressionconstruct may exist in the cell extrachromosomally or integrated intothe chromosome. When integrated into the chromosome the nucleic acidscomprising the expression vector or expression construct will remain anexpression vector or expression construct.

As used herein, the term “fusion protein” and “fusion polypeptide” areused interchangeably and both refer to two or more nucleotide sequencesobtained from different genes that have been cloned together and thatencode a single polypeptide segment. Fusion proteins are also referredto as “hybrid proteins” or “chimeric proteins.” As used herein, the term“fusion protein” includes polypeptide coding segments that are obtainedfrom different species, as well as coding segments that are obtainedfrom the same species.

As used herein, the term “heterologous” when used with reference toportions of a polynucleotide indicates that the nucleic acid comprisestwo or more subsequences that are not normally found in the samerelationship to each other in nature. For instance, a nucleic acid istypically recombinantly produced, having two or more sequences, e.g.,from unrelated genes arranged to make a new functional nucleic acid.Similarly, a “heterologous” polypeptide or protein refers to two or moresubsequences that are not found in the same relationship to each otherin nature.

As used herein, the term “host cell” refers to a prokaryotic oreukaryotic cell into which the vectors of the invention may beintroduced, expressed and/or propagated. A microbial host cell is a cellof a prokaryotic or eukaryotic microorganism, including bacteria,yeasts, microscopic fungi and microscopic phases in the life-cycle offungi and slime molds. Typical prokaryotic host cells include variousstrains of E. coli. Typical eukaryotic host cells are yeast orfilamentous fungi, or mammalian cells, such as Chinese hamster cells,murine NIH 3T3 fibroblasts, human kidney cells, or rodent myeloma orhybridoma cells.

As used herein, the term “isolated” refers to a nucleic acid orpolypeptide separated not only from other nucleic acids or polypeptidesthat are present in the natural source of the nucleic acid orpolypeptide, but also from other cellular components, and preferablyrefers to a nucleic acid or polypeptide found in the presence of (ifanything) only a solvent, buffer, ion, or other component normallypresent in a solution of the same. The terms “isolated” and “purified”do not encompass nucleic acids or polypeptides present in their naturalsource.

As used herein, the term “mammal” refers to warm-blooded vertebrateanimals all of which possess hair and suckle their young.

As used herein, the term “naturally occurring” means that the componentsare encoded by a single gene that was not altered by recombinant meansand that pre-exists in an organism.

As used herein, an “odorant” refers to any substance that can bedetected by at least one Olfactory Receptor.

As used herein, “olfaction” or “olfactory reception” refers to thedetection of compounds by an Olfactory Receptor coupled to a cellsignaling pathway. The compound detected is termed an “odorant” and maybe air-borne (i.e., volatile) and/or in solution.

As used herein, the terms “Olfactory Receptor” or “OR” are usedinterchangeably herein to refer to olfactory receptors, trace amineassociated receptors, vomeronasal receptors, formyl peptide receptors,membrane guanylyl cyclase, subtype GC-D receptors, and G-protein coupledtaste receptors. Olfactory Receptors include hybrid receptors made fromolfactory receptors, trace amine associated receptors, vomeronasalreceptors, formyl peptide receptors, membrane guanylyl cyclase, subtypeGC-D receptors, and G-protein coupled taste receptors.

As used herein, “percentage of sequence identity” and “percentagehomology” are used interchangeably herein to refer to comparisons amongpolynucleotides or polypeptides, and are determined by comparing twooptimally aligned sequences over a comparison window, where the portionof the polynucleotide or polypeptide sequence in the comparison windowmay comprise additions or deletions (i.e., gaps) as compared to thereference sequence for optimal alignment of the two sequences. Thepercentage may be calculated by determining the number of positions atwhich the identical nucleic acid base or amino acid residue occurs inboth sequences to yield the number of matched positions, dividing thenumber of matched positions by the total number of positions in thewindow of comparison and multiplying the result by 100 to yield thepercentage of sequence identity. Alternatively, the percentage may becalculated by determining the number of positions at which either theidentical nucleic acid base or amino acid residue occurs in bothsequences or a nucleic acid base or amino acid residue is aligned with agap to yield the number of matched positions, dividing the number ofmatched positions by the total number of positions in the window ofcomparison and multiplying the result by 100 to yield the percentage ofsequence identity. Those of skill in the art appreciate that there aremany established algorithms available to align two sequences. Optimalalignment of sequences for comparison can be conducted, e.g., by thelocal homology algorithm of Smith and Waterman, Adv Appl Math. 2:482,1981; by the homology alignment algorithm of Needleman and Wunsch, J MolBiol. 48:443, 1970; by the search for similarity method of Pearson andLipman, Proc Natl Acad Sci. USA 85:2444, 1988; by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe GCG Wisconsin Software Package), or by visual inspection (seegenerally, Current Protocols in Molecular Biology, F. M. Ausubel et al.,eds., Greene Publishing Associates, Inc. and John Wiley & Sons, Inc.,(1995 Supplement). Examples of algorithms that are suitable fordetermining percent sequence identity and sequence similarity are theBLAST and BLAST 2.0 algorithms, which are described in Altschul et al.,J. Mol. Biol. 215:403-410, 1990; and Altschul et al., Nucleic Acids Res.25(17):3389-3402, 1977; respectively. Software for performing BLASTanalyses is publicly available through the National Center forBiotechnology Information website. BLAST for nucleotide sequences canuse the BLASTN program with default parameters, e.g., a wordlength (W)of 11, an expectation (E) of 10, M=5, N=−4, and a comparison of bothstrands. BLAST for amino acid sequences can use the BLASTP program withdefault parameters, e.g., a wordlength (W) of 3, an expectation (E) of10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff, ProcNatl Acad Sci. USA 89:10915, 1989). Exemplary determination of sequencealignment and % sequence identity can also employ the BESTFIT or GAPprograms in the GCG Wisconsin Software package (Accelrys, Madison Wis.),using default parameters provided.

As used herein, the terms “protein”, “peptide”, “polypeptide” and“polypeptide fragment” are used interchangeably herein to refer topolymers of amino acid residues of any length. The polymer can be linearor branched, it may comprise modified amino acids or amino acid analogs,and it may be interrupted by chemical moieties other than amino acids.The terms also encompass an amino acid polymer that has been modifiednaturally or by intervention; for example disulfide bond formation,glycosylation, lipidation, acetylation, phosphorylation, PEGylation orany other manipulation or modification, such as conjugation with alabeling or bioactive component.

As used herein, the term “purified” means that the indicated nucleicacid or polypeptide is present in the substantial absence of otherbiological macromolecules, e.g., polynucleotides, proteins, and thelike. In one embodiment, the polynucleotide or polypeptide is purifiedsuch that it constitutes at least 95% by weight, more preferably atleast 99.8% by weight, of the indicated biological macromoleculespresent (but water, buffers, and other small molecules, especiallymolecules having a molecular weight of less than 1000 daltons, can bepresent).

As used herein, the term “real time” refers to taking multiplemeasurements during a reaction or interaction as opposed to making asingle measurement at the end of the reaction, or at a specified timepoint. Real time measurements are often used to quantitate the amount ofa component in a sample, or to provide relative quantification of two ormore components in a sample. Real time measurements can also be used todetermine kinetic parameters of a reaction or interaction.

As used herein, the term “recombinant nucleic acid” refers to a nucleicacid in a form not normally found in nature. For example, a recombinantnucleic acid may be flanked by a nucleotide sequence not naturallyflanking the nucleic acid or the recombinant nucleic acid may have asequence not normally found in nature. Recombinant nucleic acids can beoriginally formed in vitro by the manipulation of nucleic acid byrestriction endonucleases, or alternatively using such techniques aspolymerase chain reaction. It is understood that once a recombinantnucleic acid is made and reintroduced into a host cell or organism, itmay replicate non-recombinantly, i.e., using the in vivo cellularmachinery of the host cell rather than in vitro manipulations; however,such nucleic acids, once produced recombinantly, although subsequentlyreplicated non-recombinantly, are still considered recombinant for thepurposes of the invention.

As used herein, the term “recombinant polypeptide” refers to apolypeptide expressed from a recombinant nucleic acid, or a polypeptidethat is chemically synthesized in vitro.

As used herein, the term “recombinant variant” refers to any polypeptidediffering from naturally occurring polypeptides by amino acidinsertions, deletions, and substitutions, created using recombinant DNAtechniques. Guidance in determining which amino acid residues may bereplaced, added, or deleted without abolishing activities of interest,such as enzymatic or binding activities, may be found by comparing thesequence of the particular polypeptide with that of homologous peptidesand minimizing the number of amino acid sequence changes made in regionsof high homology.

As used herein, the terms “repertoire” or “library” refers to a libraryof genes encoding a plurality of different Olfactory Receptors. In someembodiments, the repertoire or library represents all of the OlfactoryReceptors of a species, e.g., human, dog, or cat. In some embodiments,the repertoire or library represents the Olfactory Receptors that detecta taste, scent, smell, aroma, and/or odor. In some embodiments, therepertoire or library represents the Olfactory Receptors that detect adesired, pleasing, arousing, or adverse taste, scent, smell, aroma,and/or odor. In some embodiments, the repertoire or library representsthe Olfactory Receptors of a class, family, or type.

As used herein, the term “reporter” or “reporter molecule” refers to amoiety capable of being detected indirectly or directly. Reportersinclude, without limitation, a chromophore, a fluorophore, a fluorescentprotein, a luminescent protein, a receptor, a hapten, an enzyme, and aradioisotope.

As used herein, the term “reporter gene” refers to a polynucleotide thatencodes a reporter molecule that can be detected, either directly orindirectly. Exemplary reporter genes encode, among others, enzymes,fluorescent proteins, bioluminescent proteins, receptors, antigenicepitopes, and transporters.

As used herein, “stringent hybridization conditions” refers tohybridizing in 50% formamide at 5×SSC at a temperature of 42° C. andwashing the filters in 0.2×SSC at 60° C. (1×SSC is 0.15M NaCl, 0.015Msodium citrate.) Stringent hybridization conditions also encompasses lowionic strength and high temperature for washing, for example 0.015 Msodium chloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at50° C.; hybridization with a denaturing agent, such as formamide, forexample, 50% (v/v) formamide with 0.1% bovine serum albumin/0.1%Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5with 750 mM sodium chloride, 75 mM sodium citrate at 42° C.; or 50%formamide, 5×SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodiumphosphate (pH 6.8), 0.1% sodium pyrophosphate, 5×Denhardt's solution,sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS, and 10% dextran sulfateat 42° C., with washes at 42° C. in 0.2×SSC (sodium chloride/sodiumcitrate) and 50% formamide at 55° C., followed by a high-stringency washconsisting of 0.1×SSC containing EDTA at 55° C.

As used herein, “substantial identity” refers to a polynucleotide orpolypeptide sequence that has at least 80 percent sequence identity, atleast 85 percent identity and 89 to 95 percent sequence identity.Substantial identity also encompasses at least 99 percent sequenceidentity as compared to a reference sequence over a comparison window ofat least 20 residue positions or a window of at least 30-50 residues,wherein the percentage of sequence identity is calculated by comparingthe reference sequence to a sequence that includes deletions oradditions or substitutions over the window of comparison. In specificembodiments applied to polypeptides, the term “substantial identity”means that two polypeptide sequences, when optimally aligned, such as bythe programs GAP or BESTFIT using standard parameters, i.e., defaultparameters, share at least 80 percent sequence identity, preferably atleast 89 percent sequence identity, at least 95 percent sequenceidentity or more (e.g., 99 percent sequence identity).

Preferably, amino acid “substitutions” are the result of replacing oneamino acid with another amino acid having similar structural and/orchemical properties, i.e., conservative amino acid replacements. Aminoacid substitutions may be made on the basis of similarity in polarity,charge, solubility, hydrophobicity, hydrophilicity, and/or theamphipathic nature of the residues involved. For example, nonpolar(hydrophobic) amino acids include alanine, leucine, isoleucine, valine,proline, phenylalanine, tryptophan, and methionine; polar neutral aminoacids include glycine, serine, threonine, cysteine, tyrosine,asparagine, and glutamine; positively charged (basic) amino acidsinclude arginine, lysine, and histidine; and negatively charged (acidic)amino acids include aspartic acid and glutamic acid.

As used herein, “taste receptors” refers to G-protein coupled tastereceptors for detecting sweet, bitter, and umami (glutamate), and ionchannels and ionotropic receptors for detecting salty and sour.

As used herein, “transfected” or “transformed” or “transduced” aredefined to be a process by which an exogenous nucleic acid istransferred or introduced into a host cell. A “transfected” or“transformed” or “transduced” cell is one which has been transfected,transformed or transduced with exogenous nucleic acid. The cell includesthe primary subject cell and its progeny.

The singular terms “a”, “an”, and “the” include plural referents unlesscontext clearly indicates otherwise. Similarly, the word “or” isintended to include “and” unless the context clearly indicatesotherwise. Numerical limitations given with respect to concentrations orlevels of a substance, such as an antigen, are intended to beapproximate. Thus, where a concentration is indicated to be at least(for example) 200 μg, it is intended that the concentration beunderstood to be at least approximately “about” or “about” 200 μg.

Olfactory Receptors

Most Olfactory Receptors are G-protein coupled receptors that associatewith a G-protein for signal transduction after the receptor is activatedby an odorant. GPCRs have a conserved structural feature of sevenα-helical transmembrane regions. Most olfactory receptors are about320±25 amino acids in length. The differences in length mostly resultfrom variations in the N-terminal and C-terminal regions. Most olfactoryreceptors include the motif MAYDRYVAIC (SEQ ID NO:2) located at thejunction of TM3 (transmembrane section 3) and the intracellular loopbetween TM3 and TM4. Other motifs conserved in some of the olfactoryreceptors, include, for example, LHTPMY (SEQ ID NO:3) within the firstintracellular loop, FSTCSSH (SEQ ID NO:4) at the beginning of TM6, andPMLNPF (SEQ ID NO:5) in TM7.

In some embodiments, the olfactory receptors used in the invention arehuman olfactory receptors, or olfactory receptors from another mammal,or olfactory receptors from another organism. In some embodiments,olfactory receptors used in the invention are hybrid olfactoryreceptors. In some embodiments, amino acids from the N-terminal regionof one olfactory receptor are fused to the N-terminal region of asecond, different olfactory receptor. In some embodiments, theN-terminal amino acids are from amino acid positions 1-61 of the donorolfactory receptor. In some embodiments, the N-terminal amino acids arefrom amino acid positions 1-55 of the donor olfactory receptor. In someembodiments, the N-terminal amino acids are from amino acid positions1-20 or the amino acids up to the first transmembrane domain, or aminoacid positions 1-40 which includes the consensus sequence of the firsttransmembrane domain. In some embodiments, the N-terminal amino acidsare fused to the acceptor olfactory receptor at its N-terminal region ofamino acid positions 1-61. In some embodiments, amino acids from theC-terminus of a donor polypeptide are fused to the C-terminal end of theacceptor olfactory receptor. In some embodiments, 1-50 amino acids fromthe C-terminus of the acceptor olfactory receptor are replaced by aminoacids from a donor polypeptide. In some embodiments, 1-55 amino acidsfrom the C-terminus of the acceptor olfactory receptor are replaced byamino acids from a donor polypeptide. In some embodiments, the donorpolypeptide is an olfactory receptor.

In some embodiments, the acceptor olfactory receptor is a humanolfactory receptor and the donor olfactory receptor is a human olfactoryreceptor. In some embodiments, the acceptor olfactory receptor is ahuman olfactory receptor and the donor olfactory receptor is a murineolfactory receptor. In some embodiments, the acceptor olfactory receptoris a human olfactory receptor and the donor olfactory receptor is ayeast polypeptide. In some embodiments, the acceptor olfactory receptoris a murine olfactory receptor and the donor olfactory receptor is amurine olfactory receptor. In some embodiments, the acceptor olfactoryreceptor is a murine olfactory receptor and the donor olfactory receptoris a human olfactory receptor. In some embodiments, the acceptorolfactory receptor is a murine olfactory receptor and the donorolfactory receptor is a yeast polypeptide. In some embodiments, theacceptor olfactory receptor is a canine olfactory receptor and the donorolfactory receptor is a canine olfactory receptor. In some embodiments,the acceptor olfactory receptor is a canine olfactory receptor and thedonor olfactory receptor is a human or a murine olfactory receptor. Insome embodiments, the acceptor olfactory receptor is a canine olfactoryreceptor and the donor olfactory receptor is a yeast polypeptide.

In some embodiments, the amino acids added from the donor olfactoryreceptor replace the corresponding amino acid positions in the acceptorolfactory receptor. In some embodiments, the added amino acids from thedonor olfactory receptor increase the total number of amino acids in theacceptor olfactory receptor. In some embodiments, the acceptor olfactoryreceptor has fewer amino acids (than the starting acceptor olfactoryreceptor) after the fusion is made.

Most mammalian olfactory receptors can be classified into twophylogenetic groups, class I and class II olfactory receptors. Class Iolfactory receptors are similar to fish olfactory receptors and class IIreceptors are most characteristic of mammals. In mammals a majority ofthe olfactory receptors are in class II, but mammals also have class Ireceptors, for example, humans and mice each have more than 100 class Iolfactory receptors. The number of olfactory genes varies among mammalsfrom about 800 (including pseudogenes) in primates to about 1,500 indogs and mice. The number of functional olfactory receptors varies fromabout 262 in platypus and 390 in humans to 1,284 in rats and 1,194 inmice.

The repertoire of human olfactory receptors includes about 850 genes andpseudogenes, including about 390 putatively functional genes, in 18 genefamilies, and 300 subfamilies. Databases setting out the organization ofthe human olfactory receptor genes into families and subfamilies, alongwith links to the polypeptide and nucleic acid sequences of theolfactory receptors can be found at HUGO Gene Nomenclature Committeewebsite, genenames.org/genefamilies/OR, the Olfactory Receptors Databaseat senselab.med.yale.edu/ORDB/info/humanorseqanal, and HORDE, the HumanOlfactory Data Explorer, found at genome.weizmann.ac.il/horde/, all ofwhich are incorporated by reference in their entirety for all purposes.

The repertoire of mouse olfactory receptors includes about 1,296 genesand pseudogenes, of which about 80% are putatively functional, in 228families. Databases with the organization of the mouse olfactoryreceptor genes into families and subfamilies, along with links to thepolypeptide and nucleic acid sequences of the olfactory receptors can befound at the Olfactory Receptors Database atsenselab.med.yale.edu/ORDB/info/humanorseqanal, which is incorporated byreference in its entirety for all purposes.

The repertoire of canine olfactory receptors includes about 1,094 genes.Quignon et al., Genome Biol. vol. 6, pp. R83-R83.9 (2005); Olender etal., Genomics vol. 83, pp. 361-372 (2004); Quignon et al., Chapter 13,CSH Monographs Volume 44: The Dog and Its Genome (2006); which areincorporated by reference in their entirety for all purposes.

The Olfactory Receptor repertoires of other mammals are also within thescope of the invention, including, for example, the Olfactory Receptorrepertoires of mice, rats, cats, cows and cattle, horses, goats, pigs,and bears.

In some embodiments, a biosensor is made from human olfactory receptor1A1 having the amino acid sequence (OR1A1, NCBI 9606, UP000005640, HGNC8179, NP 055380.2, DMDM 212276451):

(SEQ ID NO: 6) MRENNQSSTL EFILLGVTGQ QEQEDFFYIL FLFIYPITLIGNLLIVLAIC SDVRLHNPMY FLLANLSLVD IFFSSVTIPKMLANHLLGSK SISFGGCLTQ MYFMIALGNT DSYILAAMAYDRAVAISRPL HYTTIMSPRS CIWLIAGSWV IGNANALPHTLLTASLSFCG NQEVANFYCD ITPLLKLSCS DIHFHVKMMY LGVGIFSVPL LCIIVSYIRV FSTVFQVPST KGVLKAFSTCGSHLTVVSLY YGTVMGTYFR PLTNYSLKDA VITVMYTAVTPMLNPFIYSL RNRDMKAALR KLFNKRISS In some embodiments, N-terminal amino acids of the human olfactoryreceptor 1A1 are replaced with N-terminal amino acids from the humanolfactory receptor 6A2 having the sequence (OR6A2, NCBI 9606,UP000005640, HGNC 15301; NP 003687.2)

(SEQ ID NO: 7) MEWRNHSGRV SEFVLLGFPA PAPLQVLLFA LLLLAYVLVLTENTLIIMAI RNHSTLHKPM YFFLANMSFL EIWYVTVTIPKMLAGFVGSK QDHGQLISFE GCMTQLYFFL GLGCTECVLLAVMAYDRYMA ICYPLHYPVI VSGRLCVQMA AGSWAGGFGISMVKVFLISG LSYCGPNIIN HFFCDVSPLL NLSCTDMSTA ELTDFILAIF ILLGPLSVTG ASYVAITGAV MHIPSAAGRYKAFSTCASHL TVVIIFYAAS IFIYARPKAL SAFDTNKLVSVLYAVIVPLL NPIIYCLRNQ EVKRALCCTL HLYQHQDPDP KKASRNV In some embodiments, amino acids from the N-terminal region of OR6A2(amino acid positions 1-61) are fused to OR1A1 to make a fusionolfactory receptor to be used in the biosensor. In some embodiments, atleast 20 contiguous amino acids from the N-terminal region of OR6A2 arefused to with OR1A1. In some embodiments, the N-terminal region of OR6A2is amino acid positions 1-55. These amino acids of OR6A2 are fused at aposition in the N-terminal region of OR1A1, ranging from 1-61. In someembodiments, the N-terminal sequence from OR6A2 is fused to amino acidposition 56 of OR1A1. In some embodiments, the human OR6A2 is used inthe biosensor without modification. In some embodiments, the human OR6A2receptor is modified at its C-terminal end by fusing with otherC-terminal sequences from other olfactory receptors. In someembodiments, the human OR6A2 is modified at its N-terminal end by fusingN-terminal sequences from other olfactory receptors.

In some embodiments, a biosensor is made from the human olfactoryreceptor 2J2 (OR2J2, HGNC 8260; NP_112167).

(SEQ ID NO: 8) MMIKKNASSE DFFILLGFSN WPQLEVVLFV VILIFYLMTLTGNLFIIILS YVDSHLHTPM YFFLSNLSFL DLCYTTSSIPQLLVNLRGPE KTISYAGCMV QLYFVLALGI TECVLLVVMSYDRYVAVCRP LHYTVLMHPR FCHLLVAASW VIGFTISALHSSFTFWVPLC GHRLVDHFFC EVPALLRLSC VDTHANELTL MVMSSIFVLI PLILILTTYG AIARAVLSMQ STTGLQKVFRTCGAHLMVVS LFFIPVMCMY LQPPSENSPD QGKFIALFYTVVTPSLNPLI YTLRNKHVKG AAKRLLGWEW GK 

In some embodiments, a biosensor is made from the human olfactoryreceptor 2W1 (OR2W1, HGNC 8281; NP_112165).

(SEQ ID NO: 9) MDQSNYSSLH GFILLGFSNH PKMEMILSGV VAIFYLITLVGNTAIILASL LDSQLHTPMY FFLRNLSFLD LCFTTSIIPQMLVNLWGPDK TISYVGCIIQ LYVYMWLGSV ECLLLAVMSYDRFTAICKPL HYFVVMNPHL CLKMIIMIWS ISLANSVVLCTLTLNLPTCG NNILDHFLCE LPALVKIACV DTTTVEMSVF ALGIIIVLTP LILILISYGY IAKAVLRTKS KASQRKAMNTCGSHLTVVSM FYGTIIYMYL QPGNRASKDQ GKFLTLFYTVITPSLNPLIY TLRNKDMKDA LKKLMRFHHK STKIKRNCKS 

In some embodiments, a biosensor is made from the human olfactoryreceptor 5P3 (OR5P3, HGNC 14784; NP_703146).

(SEQ ID NO: 10) MGTGNDTTVV EFTLLGLSED TTVCAILFLV FLGIYVVTLMGNISIIVLIR RSHHLHTPMY IFLCHLAFVD IGYSSSVTPVMLMSFLRKET SLPVAGCVAQ LCSVVTFGTA ECFLLAAMAYDRYVAICSPL LYSTCMSPGV CIILVGMSYL GGCVNAWTFIGCLLRLSFCG PNKVNHFFCD YSPLLKLACS HDFTFEIIPA ISSGSIIVAT VCVIAISYIY ILITILKMHS TKGRHKAFSTCTSHLTAVTL FYGTITFIYV MPKSSYSTDQ NKVVSVFYTVVIPMLNPLIY SLRNKEIKGA LKRELRIKIF S 

In some embodiments, N-terminal amino acids from the rat RI7 olfactoryreceptor are fused to the N-terminal end of a human olfactory receptor.The rat RI7 olfactory receptor has the N-terminal sequence:

(SEQ ID NO: 11) MERRNHSGRV SEFVLLGFPA PAPLRVLLFF LSLLAYVLVLTENMLIIIAI RNHPTLHKPM YFFLANMSFL EIWYVTVTIP KMLAGFIGSK ENHGQLISFE In some embodiments, amino acid positions 1-55 of the N-terminalsequence of the rat RI7 olfactory receptor are fused to the N-terminalend of the human olfactory receptor.

In some embodiments, the Olfactory Receptor is fused at its N- orC-terminal end with FLAG or HIS tags to assist in certain purificationand biochemical characterizations of the biosensor polypeptides.

Human olfactory receptors are classified into 18 families: 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 51, 52, 55 and 56. Family OR1 has 21members: OR1A, OR1B, OR1C, OR1D, OR1E, OR1F, OR1G, OR1H, OR1I, OR1J,OR1K, OR1L, OR1M, OR1N, OR1P, OR1Q, OR1R, OR1S, OR1X, OR1AA, and OR1AB.Family OR2 has 41 members: OR2A, OR2B, OR2C, OR2D, OR2E, OR2F, OR2G,OR2H, OR21, OR2J, OR2K, OR2L, OR2M, OR2N, OR2Q, OR2R, OR2S, OR2T, OR2U,OR2V, OR2W, OR2X, OR2Y, OR2Z, OR2AD, OR2AE, OR2AF, OR2AG, OR2AH, OR2AI,OR2AJ, OR2AK, OR2AL, OR2AM, OR2AO, OR2AP, OR2AS, and OR2AT. Family OR3has 3 members: OR3A, OR3B, and OR3D. Family OR4 has 21 members: OR4A,OR4B, OR4C, OR4D, OR4E, OR4F, OR4G, OR4H, OR4K, OR4L, OR4M, OR4N, OR4P,OR4Q, OR4R, OR4S, OR4T, OR4U, OR4V, OR4W, and OR4X. Family OR5 has 49members: OR5A, OR5B, OR5C, OR5D, OR5E, OR5F, OR5G, OR5H, OR51, OR5J,OR5K, OR5L, OR5M, OR5P, OR5R, OR5S, OR5T, OR5V, OR5W, OR5AC, OR5AH,OR5AK, OR5AL, OR5AM, OR5AN, OR5AO, OR5AP, OR5AQ, OR5AR, OR5AS, OR5AU,OR5W, OR5X, OR5Y, OR5Z, OR5BA, OR5BB, OR5BC, OR5BD, OR5BE, OR5BH, OR5BJ,OR5BK, OR5BL, OR5BM, OR5BN, OR5BP, OR5BQ, OR5BR, OR5BS, and OR5BT.Family OR6 has 21 members: OR6A, OR6B, OR6C, OR6D, OR6E, OR6F, OR6J,OR6K, OR6L, OR6M, OR6N, OR6P, OR6Q, OR6R, OR6S, OR6T, OR6U, OR6V, OR6W,OR6X, and OR6Y. Family OR7 has 9 members: OR7A, OR7C, OR7D, OR7E, OR7G,OR7H, OR7K, OR7L, and OR7M. Family OR8 has 18 members: OR8A, OR8B, OR8C,OR8D, OR8F, OR8G, OR8H, OR81, OR8J, OR8K, OR8L, OR8Q, OR8R, ORBS, OR8T,OR8U, OR8V, and OR8X. Family OR9 has 12 members: OR9A, OR9G, OR9H, OR9J,OR9K, OR9L, OR9M, OR9N, OR9P, OR9Q, OR9R, and OR9S. Family OR10 has 29members: OR10A, OR10B, OR10C, OR10D, OR10G, OR10H, OR10J, OR10K, OR10N,OR10P, OR10Q, OR10R, OR10S, OR10T, OR10U, OR10V, OR10W, OR10X, OR10Y,OR10Z, OR10AA, OR10AB, OR10AC, OR10AD, OR10AE, OR10AF, OR10AG, OR10AH,and OR10AK. Family OR11 has 11 members: OR11A, OR11G, OR11H, OR11I,OR11J, OR11K, OR11L, OR11M, OR11N, OR11P, OR11Q. Family OR12 has 1member: OR12D. Family OR13 has 11 members: OR13A, OR13C, OR13D, OR13E,OR13F, OR13G, OR13H, OR131, OR13J, OR13K, and OR13Z. Family OR14 has 6members: OR14A, OR14C, OR141, OR14J, OR14K, and OR14L. Family OR51 has21 members: OR51A, OR51B, OR51C, OR51D, OR51E, OR51F, OR51G, OR51H,OR511, OR51J, OR51K, OR51L, OR51M, OR51N, OR51P, OR51Q, OR51R, OR51S,OR51T, OR51V, and OR51AB. Family OR52 has 22 members: OR52A, OR52B,OR52D, OR52E, OR52H, OR521, OR52J, OR52K, OR52L, OR52M, OR52N, OR52P,OR52Q, OR52R, OR52S, OR52T, OR52U, OR52V, OR52W, OR52X, OR52Y, andOR52Z. Family OR55 has 1 member: OR55B. Family OR56 has 2 members: OR56Aand OR56B.

Biosensors

The invention relates to biosensors for the detection of interactions atan Olfactory Receptor. In some embodiments, the biosensors are used todetect the interaction of an odorant at an Olfactory Receptor. In someembodiments, a biosensor comprises a plurality of Olfactory Receptorsand the plurality of Olfactory Receptors are used to detect an odorant.In some embodiments, the plurality of Olfactory Receptors in thebiosensor represent the repertoire or a portion of the repertoire of ananimals Olfactory Receptors. In some embodiments, the plurality ofOlfactory Receptors in the biosensor represents the repertoire or aportion of the repertoire of human Olfactory Receptors. In someembodiments, the plurality of Olfactory Receptors in the biosensorrepresents the portion of the repertoire of human Olfactory Receptorsthat detect odorants in solution. In some embodiments, the plurality ofOlfactory Receptors in the biosensor represents the portion of therepertoire of human Olfactory Receptors that detect odorants in thegaseous phase. In some embodiments, the plurality of Olfactory Receptorsin the biosensor represents the portion of the repertoire of humanOlfactory Receptors that produce a pleasurable or positive response. Insome embodiments, the plurality of Olfactory Receptors in the biosensorrepresents the portion of the repertoire of human Olfactory Receptorsthat produce an adverse or negative response. In some embodiments, theplurality of Olfactory Receptors in the biosensor represents the portionof the repertoire of human Olfactory Receptors from one of the 18families of human Olfactory Receptors.

In some embodiments, individual biosensors are comprised of an OlfactoryReceptor that is fused in its N-terminal region to a polypeptidesequence that targets the nascent polypeptide to the host cell secretoryapparatus for insertion of the Olfactory Receptor into the membrane, andfused in its C-terminal region to a polypeptide that stabilizes thereceptor in the membrane. In some embodiments, the polypeptide fused tothe C-terminal region of the Olfactory Receptor targets the receptor tothe outermembrane of the host cell. In some embodiments, the OlfactoryReceptor is a mammalian olfactory receptor. In some embodiments, theOlfactory Receptor is a human Olfactory Receptor. In some embodiments, afull length Olfactory Receptor is used in the biosensor. In someembodiments, the full length Olfactory Receptor is a human OlfactoryReceptor.

In some embodiments, the biosensor includes a G-protein signalingpathway. Many G-protein signaling pathways may be used. In someembodiments, the G-protein signaling pathway comprises theG-protein-mediated activation of adenylate cyclase with resultantproduction of cAMP as a second messenger. In some embodiments, the cAMPinteracts with a cAMP activated cation channel.

In some embodiments, the biosensors of the invention are also comprisedof a G-protein and an adenylate cyclase (e.g., Uniprot O60266). In someembodiments, the G-protein is comprised of three subunits the Gα subunit(e.g., Uniprot P38405), Gβ subunit (e.g., Uniprot P62879) and Gγ subunit(e.g., Uniprot P63218). In some embodiments, the adenylate cyclase andthe G protein are from the same species. In some embodiments, theadenylate cyclase and the G protein are from different species. In someembodiments, the G protein subunits from the same or from differentspecies. In some embodiments, the Olfactory Receptor, G protein andadenylate cyclase are from the same species, and in some embodiments,one or more of the components are from different species. In someembodiments, the Olfactory Receptor and G protein of the biosensororiginate from human polypeptides. In some embodiments, the biosensorpolypeptides of the invention include polypeptides that have 70%, 80%,90%, 95%, and 99% sequence homology with SEQ ID NO: 6-10 and 12-17, orone of the human OR receptors from the 18 human OR gene families.

In some embodiments, the biosensor includes a reporter. In someembodiments, the G proteins of the biosensor interact directly with areporter polypeptide to produce a detectable signal, e.g., adenylatecyclase is a reporter polypeptide that produces cAMP. The cAMP moleculeitself can be detected (e.g., commercially available kits are sold by,for example, Thermofisher Scientific, Ray Biotech, Enzo Life Sciences,Cayman Chemical, and Cell BioLabs). In some embodiments, the G proteinsof the biosensor interact with a polypeptide that induces a reporter. Insome embodiments, the G proteins interact with a polypeptide (e.g.,adenylate cyclase) to create a first signal, and a second systemamplifies the first signal when the reporter responds to the firstsignal. In some embodiments, multiple amplification steps are used toincrease detection of interactions at the Olfactory Receptor. In someembodiments, both the primary signal and the amplified or multipleamplified signals are detected so as to increase the dynamic range ofbinding interactions detected by the biosensor.

In some embodiments, the biosensor includes one or more reporters. Insome embodiments, a heterologous gene encoding a reporter protein isintroduced into the host cell such that the host cell expresses thereporter, and the biosensor activates the reporter when an appropriateinteraction occurs at the Olfactory Receptor of the biosensor. In someembodiments, the host cells are engineered to express a single reporter.In some embodiments, different host cells, each expressing a differentreporter, are used to enhance signal detection of the biosensor. In someembodiments, the host cell is engineered to express two or more reporterproducts, for example by using a single vector construct encoding two ormore reporters. In some embodiments, the reporter or reporters provide adynamic range of detection over at least 3, 4, 5, 6, 7, 8, 9, or 10orders of magnitude, covering a range of detection at OlfactoryReceptors in the range of from about 10⁻¹²M to about 10 M.

In some embodiments, the reporter or reporters are one or more of afluorescent reporter, a bioluminescent reporter, an enzyme, and an ionchannel. Examples of fluorescent reporters include, for example, greenfluorescent protein from Aequorea victoria or Renilla reniformis, andactive variants thereof (e.g., blue fluorescent protein, yellowfluorescent protein, cyan fluorescent protein, etc.); fluorescentproteins from Hydroid jellyfishes, Copepod, Ctenophora, Anthrozoas, andEntacmaea quadricolor, and active variants thereof; andphycobiliproteins and active variants thereof. Other fluorescentreporters include, for example, small molecules such as CPSD (Disodium3-(4-methoxyspiro {1,2-dioxetane-3,2′-(5′-chloro)tricyclo[3.3.1.1^(3,7)]decan}-4-yl)phenyl phosphate, ThermoFisher Catalog#T2141). Bioluminescent reporters include, for example, aequorin (andother Ca⁺² regulated photoproteins), luciferase based on luciferinsubstrate, luciferase based on Coelenterazine substrate (e.g., Renilla,Gaussia, and Metridina), and luciferase from Cypridina, and activevariants thereof. In some embodiments, the bioluminescent reporterinclude, for example, North American firefly luciferase, Japanesefirefly luciferase, Italian firefly luciferase, East European fireflyluciferase, Pennsylvania firefly luciferase, Click beetle luciferase,railroad worm luciferase, Renilla luciferase, Gaussia luciferase,Cypridina luciferase, Metrida luciferase, OLuc, and red fireflyluciferase, all of which are commercially available from ThermoFisherScientific and/or Promega. Enzyme reporters include, for example,β-galactosidase, chloramphenicol acetyltransferase, horseradishperoxidase, alkaline phosphatase, acetylcholinesterase, and catalase.Ion channel reporters, include, for example, cAMP activated cationchannels. The reporter or reporters may also include a Positron EmissionTomography (PET) reporter, a Single Photon Emission Computed Tomography(SPECT) reporter, a photoacoustic reporter, an X-ray reporter, and anultrasound reporter.

In some embodiments, antibodies are used to amplify the signal from theOlfactory Receptor binding interaction. In some embodiments, thereporter is a polypeptide or small molecule detectable by an antibody.In some embodiments, the small molecule or polypeptide is detected in anELISA. In some embodiments, an antibody sandwich assay is used toamplify the signal from the small molecule or polypeptide reporter.

In some embodiments, real time measurements are made with the biosensorof the invention. In some embodiments, the reporter emits light orproduces a molecule that can be detected with an optical sensor. Inthese embodiments, real time measurements can be obtained from thebiosensor by recording the change in light emission over time as thebiosensor interacts with a potential ligand. The real time measurementscan be used to quantify the binding interaction by an absolutemeasurement or a relative measurement. In the absolute measurement, thereal time signal is compared to a standard to determine the bindingactivity at the Olfactory Receptor. In some embodiments, known amountsof ligand to an Olfactory Receptor are used to generate a standardbinding curve for receptor occupancy versus reporter gene output.Binding of a test ligand can then be compared to the standard curve toquantify interaction of the test ligand at the Olfactory Receptor. Inthe relative measurement, the biosensor includes internal referencesthat allow differences in interactions at an Olfactory Receptor to becompared. In some embodiments, a reference G protein coupled receptor isincluded in the host cell, and a known amount of the reference ligand isadded to the reference receptor to act as a standard. In someembodiments, the reference receptor is coupled to a different reporter,e.g, a reporter polypeptide that provides a different optical signalfrom the Olfactory Receptor reporter. In some embodiments, the referenceand test receptors are coupled to different fluorescent protein such asgreen fluorescent protein, GFP, and red fluorescent protein, RFP. Theratio of green fluorescence to red fluorescence could be compared fordifferent test ligands at the same Olfactory Receptor, or to comparebinding of the same test ligand to different Olfactory Receptors.

In some embodiments, real time data is obtained from a biosensor with anon-optical reporter. In some embodiments, the signal from a firstreporter system is amplified by a second reporter system so as toincrease the signal from weak interactions at an Olfactory Receptor. Insome embodiments, the GTP/GDP ratio of the biosensor is controlled toregulate the sensitivity of the G-protein coupled signal transductionfrom the receptor. In some embodiments, the GTP/GDP ratio is controlledto alter the dynamic range of the biosensor.

The product of the reporter gene can be detected by any appropriatedetection method and apparatus, depending on the type of reporterproduct expressed from the reporter gene. By way of example, anexemplary reporter gene encodes a light producing protein (e.g.,luciferase or eGFP), and this phenotype can be detected using opticalimaging. In the descriptions herein, expression of a reporter is meantto include expression of the corresponding reporter gene resulting inexpression of the encoded reporter or reporter molecule.

In an embodiment, the polypeptides of the invention include polypeptidesencoded by nucleic acids that hybridize under stringent hybridizationconditions to nucleic acids encoding one of the polypeptides of SEQ IDNOS: 6-10 and 12-17, or encoding one of the human OR receptors from the18 families of human olfactory receptors. In an embodiment, thepolypeptides of the invention include polypeptides encoded by nucleicacids that hybridize under stringent hybridization conditions to nucleicacids encoding one of the polypeptides of SEQ ID NOS: 6-10 and 12-17, ora nucleic acid encoding one of the human OR receptors from the 18families of human olfactory receptors. In an embodiment, thepolypeptides of the invention include polypeptides encoded by nucleicacids that hybridize under stringent hybridization conditions to nucleicacids encoding the polypeptide of SEQ ID NO: 6 or 7.

In an embodiment, the polypeptides of the invention have at least 70%,80%, 90%, 95%, or 99% sequence identity to one of SEQ ID NOS: 6-10 and12-17, or one of the human OR receptors from the 18 families of humanolfactory receptors. In an embodiment, the polypeptides of the inventionhave at least 70%, 80%, 90%, 95%, or 99% sequence identity to one of SEQID NOS: 6-7. In an embodiment, the EphA3 polypeptides of the inventionhave at least 70%, 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:6.

In some embodiments, the threshold of detection of human OlfactoryReceptors in the biosensors of the invention are from about 0.15 partsper billion to about 420,000 parts per billion or over a range of 6-7orders of magnitude. In some embodiments, the range of detection ofhuman Olfactory Receptors in the biosensors of the invention are fromabout 10⁻⁹ M to about 10⁻³ M or over a range of about 6 orders ofmagnitude. In some embodiments, the range of detection is over 3, 4, 5,6, 7, 8, 9, or 10 orders of magnitude in the range of ligand from 10 Mto 10⁻¹² M.

The polypeptides of the invention encompass fragments and variants ofthe polypeptides of the invention. Thus, the term “fragment or variantpolypeptide” further contemplates deletions, additions and substitutionsto the sequence, so long as the polypeptide functions as describedherein. The term “conservative variation” denotes the replacement of anamino acid residue by another biologically similar residue, or thereplacement of a nucleotide in a nucleic acid sequence such that theencoded amino acid residue does not change or is changed to anotherstructurally, chemically or otherwise functionally similar residue. Inthis regard, particularly preferred substitutions will generally beconservative in nature, i.e., those substitutions that take place withina family of amino acids. For example, amino acids are generally dividedinto four families: (1) acidic-aspartate and glutamate; (2)basic-lysine, arginine, histidine; (3) non-polar-alanine, valine,leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and(4) uncharged polar-glycine, asparagine, glutamine, cysteine, serine,threonine, and tyrosine. Phenylalanine, tryptophan, and tyrosine aresometimes classified as aromatic amino acids. Examples of conservativevariations include the substitution of one hydrophobic residue such asisoleucine, valine, leucine or methionine for another hydrophobicresidue, or the substitution of one polar residue for another polarresidue, such as the substitution of arginine for lysine, glutamic acidfor aspartic acid, or glutamine for asparagine, and the like; or asimilar conservative replacement of an amino acid with a structurallyrelated amino acid that will not have a major effect on the biologicalactivity. Proteins having substantially the same amino acid sequence asthe reference molecule but possessing minor amino acid substitutionsthat do not substantially affect the immunogenicity of the protein are,therefore, within the definition of the reference polypeptide. All ofthe polypeptides produced by these modifications are included herein.The term “conservative variation” also includes the use of a substitutedamino acid in place of an unsubstituted parent amino acid provided thatantibodies raised to the substituted polypeptide also immunoreact withthe unsubstituted polypeptide.

“Variant” polypeptides or nucleic acids of the invention encompasspolypeptides or nucleic acids with substantially similar sequences. Forpolynucleotides, a variant comprises a deletion and/or addition of oneor more nucleotides at one or more sites within the nativepolynucleotide and/or a substitution of one or more nucleotides at oneor more sites in the native polynucleotide. As used herein, a “native”polynucleotide or polypeptide comprises a naturally occurring nucleotidesequence or amino acid sequence, respectively. Variants of a particularpolynucleotide of the invention (i.e., the reference polynucleotide) canalso be evaluated by comparison of the percent sequence identity betweenthe polypeptide encoded by a variant polynucleotide and the polypeptideencoded by the reference polynucleotide. “Variant” protein is intendedto mean a protein derived from the native protein by deletion oraddition of one or more amino acids at one or more sites in the nativeprotein and/or substitution of one or more amino acids at one or moresites in the native protein. Variant proteins encompassed by the presentinvention are biologically active, that is they possess the ability toelicit an immune response.

Homologs of polypeptides of the invention from other alleles areintended to be within the scope of the present invention. As usedherein, the term “homologs” includes analogs and paralogs. The term“anologs” refers to two polynucleotides or polypeptides that have thesame or similar function, but that have evolved separately in unrelatedhost organisms. The term “paralogs” refers to two polynucleotides orpolypeptides that are related by duplication within a genome. Paralogsusually have different functions, but these functions may be related.Analogs and paralogs of a wild-type polypeptide can differ from thewild-type polypeptide by post-translational modifications, by amino acidsequence differences, or by both. In particular, homologs of theinvention will generally exhibit at least 80-85%, 85-90%, 90-95%, or95%, 96%, 97%, 98%, 99% sequence identity, with all or part of thewild-type polypeptide or polynucleotide sequences, and will exhibit asimilar function. Variants include allelic variants. The term “allelicvariant” refers to a polynucleotide or a polypeptide containingpolymorphisms that lead to changes in the amino acid sequences of aprotein and that exist within a natural population (e.g., a virusspecies or variety). Such natural allelic variations can typicallyresult in 1-5% variance in a polynucleotide or a polypeptide. Allelicvariants can be identified by sequencing the nucleic acid sequence ofinterest in a number of different species, which can be readily carriedout by using hybridization probes to identify the same genetic locus inthose species. Any and all such nucleic acid variations and resultingamino acid polymorphisms or variations that are the result of naturalallelic variation and that do not alter the functional activity of thegene of interest, are intended to be within the scope of the invention.

As used herein, the term “derivative” or “variant” refers to apolypeptide, or a nucleic acid encoding a polypeptide, that has one ormore conservative amino acid variations or other minor modificationssuch that (1) the corresponding polypeptide has substantially equivalentfunction when compared to the wild type polypeptide or (2) an antibodyraised against the polypeptide that is immunoreactive with the wild-typepolypeptide. These variants or derivatives include polypeptides havingminor modifications of the polypeptide primary amino acid sequences thatmay result in peptides which have substantially equivalent activity ascompared to the unmodified counterpart polypeptide. Such modificationsmay be deliberate, as by site-directed mutagenesis, or may bespontaneous. The term “variant” further contemplates deletions,additions and substitutions to the sequence, so long as the polypeptidefunctions to produce an immunological response as defined herein. Theterm “variant” also includes the modification of a polypeptide where thenative signal peptide is replaced with a heterologous signal peptide tofacilitate the expression or secretion of the polypeptide from a hostspecies. The term “variant” may also include ‘mimitopes’, which arecompletely different protein sequence but similar structure, that alsoinduce cross-reactive immunity.

Polypeptides of the invention also may include amino acid sequences forintroducing a glycosylation site or other site for modification orderivatization of the polypeptide. In an embodiment, the polypeptides ofthe invention described above may include the amino acid sequence N-X-Sor N-X-T that can act as a glycosylation site. During glycosylation, anoligosaccharide chain is attached to asparagine (N) occurring in thetripeptide sequence N-X-S or N-X-T, where X can be any amino acid exceptPro. This sequence is called a glycosylation sequence. Thisglycosylation site may be placed at the N-terminus, C-terminus, orwithin the internal sequence of the polypeptide.

Host Cells

In the present invention, various eukaryotic cells can be used as thehost cell. In some embodiments, the host cell is a fungal cell, animalcell, plant cell, or algae cell. In some embodiments, the eukaryoticcells are fungi cells, including, but not limited to, fungi of thegenera Aspergillus, Trichoderma, Saccharomyces, Chrysosporium,Klyuveromyces, Candida, Pichia, Debaromyces, Hansenula, Yarrowia,Zygosaccharomyces, Schizosaccharomyces, Penicillium, or Rhizopus. Insome embodiments, the fungi cells are Saccharomyces cerevisiae, Pichiapastoris, Aspergillus niger, Aspergillus oryzae, Chrysosporiumlucknowense, or Trichoderma reesei.

In some embodiments, the host cells of the invention are animal cells.In some embodiments, the host cells are cells from a commerciallyvaluable livestock. In some embodiments, the animal cells are mammaliancells, such as that of bovine, canine, feline, hamster, mouse, porcine,rabbit, rat, or sheep. In some embodiments, the mammalian cells arecells of primates, including but not limited to, monkeys, chimpanzees,gorillas, and humans. In some embodiments, the mammalians cells aremouse cells, as mice routinely function as a model for other mammals,most particularly for humans (see, e.g., Hanna, J. et al., “Treatment ofsickle cell anemia mouse model with iPS cells generated from autologousskin,” Science 318:1920-23, 2007; Holtzman, D. M. et al., “Expression ofhuman apolipoprotein E reduces amyloid-β deposition in a mouse model ofAlzheimer's disease,” J Clin Invest. 103(6):R15-R21, 1999; Warren, R. S.et al., “Regulation by vascular endothelial growth factor of human coloncancer tumorigenesis in a mouse model of experimental liver metastasis,”J Clin Invest. 95: 1789-1797, 1995; each publication incorporated hereinby reference). Animal cells include, for example, fibroblasts,epithelial cells (e.g., renal, mammary, prostate, lung), keratinocytes,hepatocytes, adipocytes, endothelial cells, hematopoietic cells. In someembodiments, the animal cells are adult cells (e.g., terminallydifferentiated, dividing or non-dividing) or stem cells. In someembodiments, mammalian cell lines are used as host cells of theinvention. In some embodiments, the cell lines are derived from Chinesehamster cells, Human kidney cells, Monkey kidney cells, Human cervicalcancer cells, or Mouse myeloma cells. These and other mammalian celllines are well known in the art, for example, the mammalian cell linespublicly available from ThermoFisher Scientific, ATCC (American TypeCulture Collection), and Charles River Laboratories International, Inc.The cell lines disclosed at the web-sites for ThermoFisher, ATCC, andCharles River Laboratories are incorporate by reference in theirentirety for all purposes.

In some embodiments the eukaryotic cells are plant cells. In someembodiments the plant cells are cells of monocotyledonous ordicotyledonous plants, including, but not limited to, alfalfa, almonds,asparagus, avocado, banana, barley, bean, blackberry, brassicas,broccoli, cabbage, canola, carrot, cauliflower, celery, cherry, chicory,citrus, coffee, cotton, cucumber, eucalyptus, hemp, lettuce, lentil,maize, mango, melon, oat, papaya, pea, peanut, pineapple, plum, potato(including sweet potatoes), pumpkin, radish, rapeseed, raspberry, rice,rye, sorghum, soybean, spinach, strawberry, sugar beet, sugarcane,sunflower, tobacco, tomato, turnip, wheat, zucchini, and other fruitingvegetables (e.g. tomatoes, pepper, chili, eggplant, cucumber, squashetc.), other bulb vegetables (e.g., garlic, onion, leek etc.), otherpome fruit (e.g. apples, pears etc.), other stone fruit (e.g., peach,nectarine, apricot, pears, plums etc.), Arabidopsis, woody plants suchas coniferous and deciduous trees, an ornamental plant, a perennialgrass, a forage crop, flowers, other vegetables, other fruits, otheragricultural crops, herbs, grass, or perennial plant parts (e.g., bulbs;tubers; roots; crowns; stems; stolons; tillers; shoots; cuttings,including un-rooted cuttings, rooted cuttings, and callus cuttings orcallus-generated plantlets; apical meristems etc.). The term “plants”refers to all physical parts of a plant, including seeds, seedlings,saplings, roots, tubers, stems, stalks, foliage and fruits.

In some embodiments, the eukaryotic cells are algal, including but notlimited to algae of the genera Chlorella, Chlamydomonas, Scenedesmus,Isochrysis, Dunaliella, Tetraselmis, Nannochloropsis, or Prototheca,

Nucleic Acids

In some embodiments, the present invention relates to the nucleic acidsthat encode, at least in part, the individual peptides, polypeptides,and proteins of the present invention. In some embodiments, the nucleicacids may be natural, synthetic or a combination thereof. The nucleicacids of the invention may be RNA, mRNA, DNA, cDNA, or synthetic nucleicacids.

In some embodiments, the nucleic acids of the invention also includeexpression vectors, such as plasmids, or viral vectors, or linearvectors, or vectors that integrate into chromosomal DNA. Expressionvectors can contain a nucleic acid sequence that enables the vector toreplicate in one or more selected host cells. Such sequences are wellknown for a variety of cells. The origin of replication from the plasmidpBR322 is suitable for most Gram-negative bacteria. In eukaryotic hostcells, e.g., mammalian cells, the expression vector can be integratedinto the host cell chromosome and then replicate with the hostchromosome. Similarly, vectors can be integrated into the chromosome ofprokaryotic cells. In some embodiments, the vector is related to theautonomously replicating plasmids in yeast YRp, YEp, and YCp. All threeare S. cerevisiae/E. coli shuttle vectors that typically carry amultiple cloning site (MCS) for the insertion of expression cassettes.In some embodiments, the yeast epitope tagging vectors, pESC are used.The pESC vectors are commercially available from Agilent Technologies.

Expression vectors also generally contain a selection gene, also termeda selectable marker. Selectable markers are well-known in the art forprokaryotic and eukaryotic cells, including host cells of the invention.Generally, the selection gene encodes a protein necessary for thesurvival or growth of transformed host cells grown in a selectiveculture medium. Host cells not transformed with the vector containingthe selection gene will not survive in the culture medium. Typicalselection genes encode proteins that (a) confer resistance toantibiotics or other toxins, e.g., ampicillin, neomycin, methotrexate,or tetracycline, (b) complement auxotrophic deficiencies, or (c) supplycritical nutrients not available from complex media, e.g., the geneencoding D-alanine racemase for Bacilli. In some embodiments, anexemplary selection scheme utilizes a drug to arrest growth of a hostcell. Those cells that are successfully transformed with a heterologousgene produce a protein conferring drug resistance and thus survive theselection regimen. Other selectable markers for use in bacterial oreukaryotic (including mammalian) systems are well-known in the art.Examples of yeast selection genes, include, URA3, TRP1, LEU2, HIS3,LYS2, ADE2, MET15, hphNT1, and natNT2. Da Silva et al., FEMS YeastResearch 12:197-214 (2012), which is incorporated by reference in itsentirety for all purposes. In some embodiments, these yeast selectiongenes are used with appropriate auxotrophic yeast strains.

Inducible promoters are also contemplated as part of the invention.Examples of inducible promoters include, but are not limited to yeastpromoters for GALI, GAL7, and GAL10 (galactose-inducible) CUP1 (copperion inducible), ADH2 (glucose repression), and mammalian promoters suchas a metallothionein promoter, a glucocorticoid promoter, a progesteronepromoter, a c-fos promoter, the T-REx system of ThermoFisher whichplaces expression from the human cytomegalovirus immediate-earlypromoter under the control of tetracycline operator(s), and RheoSwitchpromoters of Intrexon. Karzenowski, D. et al., BioTechiques 39:191-196(2005); Dai, X. et al., Protein Expr. Purif 42:236-245 (2005); Palli, S.R. et al., Eur. J. Biochem. 270:1308-1515 (2003); Dhadialla, T. S. etal., Annual Rev. Entomol. 43:545-569 (1998); Kumar, M. B, et al., J.Biol. Chem. 279:27211-27218 (2004); Verhaegent, M. et al., Annal. Chem.74:4378-4385 (2002); Katalam, A. K., et al., Molecular Therapy 13:S103(2006); and Karzenowski, D. et al., Molecular Therapy 13:S194 (2006), DaSilva et al., FEMS Yeast Research 12:197-214 (2012); U.S. Pat. Nos.8,895,306, 8,822,754, 8,748,125, 8,536,354, all of which areincorporated by reference in their entirety for all purposes.

Expression vectors of the invention typically have promoter elements,e.g., enhancers, to regulate the frequency of transcriptionalinitiation. Typically, these are located in the region 30-110 bpupstream of the start site, although a number of promoters have beenshown to contain functional elements downstream of the start site aswell. The spacing between promoter elements frequently is flexible, sothat promoter function is preserved when elements are inverted or movedrelative to one another. In the thymidine kinase (tk) promoter, thespacing between promoter elements can be increased to 50 bp apart beforeactivity begins to decline. Depending on the promoter, it appears thatindividual elements can function either cooperatively or independentlyto activate transcription.

The nucleic acid of the present invention can be operably linked toanother nucleic acid so as to be expressed under control of a suitablepromoter. The nucleic acid of the present invention can be also operablylinked to, in order to attain efficient transcription of the nucleicacid, other regulatory elements that cooperate with a promoter or atranscription initiation site, for example, a nucleic acid comprising anenhancer sequence, a polyA site, or a terminator sequence. In additionto the nucleic acid of the present invention, a gene that can be amarker for confirming expression of the nucleic acid (e.g. a drugresistance gene, a gene encoding a reporter enzyme, or a gene encoding afluorescent protein) may be incorporated.

In some embodiments, it may be desirable to modify the polypeptides ofthe present invention. One of skill will recognize many ways ofgenerating alterations in a given nucleic acid construct to generatevariant polypeptides Such well-known methods include site-directedmutagenesis, PCR amplification using degenerate oligonucleotides,exposure of cells containing the nucleic acid to mutagenic agents orradiation, chemical synthesis of a desired oligonucleotide (e.g., inconjunction with ligation and/or cloning to generate large nucleicacids) and other well-known techniques (see, e.g., Gillam and Smith,Gene 8:81-97, 1979; Roberts et al., Nature 328:731-734, 1987, which isincorporated by reference in its entirety for all purposes). In someembodiments, the recombinant nucleic acids encoding the polypeptides ofthe invention are modified to provide preferred codons which enhancetranslation of the nucleic acid in a selected organism.

The polynucleotides of the invention also include polynucleotidesincluding nucleotide sequences that are substantially equivalent to thepolynucleotides of the invention. Polynucleotides according to theinvention can have at least about 80%, more typically at least about90%, and even more typically at least about 95%, sequence identity to apolynucleotide of the invention. The invention also provides thecomplement of the polynucleotides including a nucleotide sequence thathas at least about 80%, more typically at least about 90%, and even moretypically at least about 95%, sequence identity to a polynucleotideencoding a polypeptide recited above. The polynucleotide can be DNA(genomic, cDNA, amplified, or synthetic) or RNA. Methods and algorithmsfor obtaining such polynucleotides are well known to those of skill inthe art and can include, for example, methods for determininghybridization conditions which can routinely isolate polynucleotides ofthe desired sequence identities.

Nucleic acids which encode protein analogs or variants in accordancewith this invention (i.e., wherein one or more amino acids are designedto differ from the wild type polypeptide) may be produced using sitedirected mutagenesis or PCR amplification in which the primer(s) havethe desired point mutations. For a detailed description of suitablemutagenesis techniques, see Sambrook et al., Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. (1989) and/or Current Protocols in Molecular Biology,Ausubel et al., eds, Green Publishers Inc. and Wiley and Sons, N.Y(1994), each of which is incorporated by reference in its entirety forall purposes. Chemical synthesis using methods well known in the art,such as that described by Engels et al., Angew Chem Intl Ed. 28:716-34,1989 (which is incorporated by reference in its entirety for allpurposes), may also be used to prepare such nucleic acids.

In some embodiments, amino acid “substitutions” for creating variantsare preferably the result of replacing one amino acid with another aminoacid having similar structural and/or chemical properties, i.e.,conservative amino acid replacements. Amino acid substitutions may bemade on the basis of similarity in polarity, charge, solubility,hydrophobicity, hydrophilicity, and/or the amphipathic nature of theresidues involved. For example, nonpolar (hydrophobic) amino acidsinclude alanine, leucine, isoleucine, valine, proline, phenylalanine,tryptophan, and methionine; polar neutral amino acids include glycine,serine, threonine, cysteine, tyrosine, asparagine, and glutamine;positively charged (basic) amino acids include arginine, lysine, andhistidine; and negatively charged (acidic) amino acids include asparticacid and glutamic acid.

“Insertions” or “deletions” are typically in the range of about 1 to 5amino acids. The variation allowed may be experimentally determined bysystematically making insertions, deletions, or substitutions of aminoacids in a polypeptide molecule using recombinant DNA techniques andassaying the resulting recombinant variants for activity.

Alternatively, recombinant variants encoding these same or similarpolypeptides may be synthesized or selected by making use of the“redundancy” in the genetic code. Various codon substitutions, such asthe silent changes which produce various restriction sites, may beintroduced to optimize cloning into a plasmid or viral vector orexpression in a particular prokaryotic or eukaryotic system. Mutationsin the polynucleotide sequence may be reflected in the polypeptide ordomains of other peptides added to the polypeptide to modify theproperties of any part of the polypeptide, to change characteristicssuch as ligand-binding affinities, or degradation/turnover rate.

Alternatively, where alteration of function is desired, insertions,deletions or non-conservative alterations can be engineered to producealtered polypeptides or chimeric polypeptides. Such alterations can, forexample, alter one or more of the biological functions or biochemicalcharacteristics of the polypeptides of the invention. For example, suchalterations may change polypeptide characteristics such asligand-binding affinities or degradation/turnover rate. Further, suchalterations can be selected so as to generate polypeptides that arebetter suited for expression, scale up and the like in the host cellschosen for expression.

In a preferred method, polynucleotides encoding the novel nucleic acidsare changed via site-directed mutagenesis. This method usesoligonucleotide sequences that encode the polynucleotide sequence of thedesired amino acid variant, as well as a sufficient adjacent nucleotideon both sides of the changed amino acid to form a stable duplex oneither side of the site of being changed. In general, the techniques ofsite-directed mutagenesis are well known to those of skill in the art,and this technique is exemplified by publications such as, Edelman etal., DNA 2:183 (1983). A versatile and efficient method for producingsite-specific changes in a polynucleotide sequence is described inZoller and Smith, Nucleic Acids Res. 10:6487-6500 (1982).

PCR may also be used to create amino acid sequence variants of thenucleic acids. When small amounts of template DNA are used as startingmaterial, primer(s) that differs slightly in sequence from thecorresponding region in the template DNA can generate the desired aminoacid variant. PCR amplification results in a population of product DNAfragments that differ from the polynucleotide template encoding thetarget at the position specified by the primer. The product DNAfragments replace the corresponding region in the plasmid and this givesthe desired amino acid variant.

A further technique for generating amino acid variants is the cassettemutagenesis technique described in Wells et al., Gene 34:315 (1985), andother mutagenesis techniques well known in the art, such as, forexample, the techniques in Sambrook et al., supra, and Ausubel et al.,supra.

Process for Making Host Cells with Biosensors

As described above, Olfactory Receptors are genetically engineered forexpression in a desired host cell. The Olfactory Receptors may be from acertain species, or maybe fusion or hybrid constructs. The N-terminaland C-terminal sequences of the Olfactory Receptor or fusion/hybridlocalize the Olfactory Receptor or fusion/hybrid to the host cellmembrane, and if appropriate to the outer membrane of a host cell. TheseOlfactory Receptor or fusion/hybrid gene constructs are placed intoappropriate expression vectors for the host cell and then theseexpression constructs or expression vectors are placed inside a hostcell.

In some embodiments, the host cells are also genetically engineered toexpress human G protein subunits. In some embodiments, the host cellsare also genetically engineered to express the human G protein subunitsGα, Gβ, and Gγ. In this embodiment, the genes encoding the human Gα, Gβ,and Gγ subunits are placed under the control of appropriate controlsequences (promoters, enhancers, translation start sequences, polyAsites, etc.) for the desired host cell, and these constructs for thehuman Gα, Gβ, and Gγ subunits are placed into the desired host cell. Insome embodiments, the human G protein is also associated with adenylatecyclase. In this embodiment, the gene for an appropriate adenylatecyclase is placed under the control of appropriate control sequences forthe desired host cell, and this construct is placed into the desiredhost cell.

In the process of the present invention, a eukaryotic host cell asdescribe above is used. In some embodiments, a fungal cell is used. Insome embodiments, the fungal cell is from the Aspergillus, Trichoderma,Saccharomyces, Chrysosporium, Klyuveromyces, Candida, Pichia,Debaromyces, Hansenula, Yarrowia, Zygosaccharomyces,Schizosaccharomyces, Penicillium, or Rhizopus genera. In someembodiments, the fungal cell is a Saccharomyces cerevisiae. In someembodiments, a eukaryotic cell derived from a mammal, for example, ahuman cell, or a cell derived from a non-human mammal such as a monkey,a mouse, a rat, a pig, a horse, or a dog can be used. The cell used inthe process of the present invention is not particularly limited, andany cell can be used.

In some embodiments, the nucleic acid encoding the biosensor isintroduced to the host cell by transfection (e.g., Gorman, et al. Proc.Natl. Acad. Sci. 79.22 (1982): 6777-6781, which is incorporated byreference in its entirety for all purposes), transduction (e.g., Cepkoand Pear (2001) Current Protocols in Molecular Biology unit 9.9; DOI:10.1002/0471142727.mb0909s36, which is incorporated by reference in itsentirety for all purposes), calcium phosphate transformation (e.g.,Kingston, Chen and Okayama (2001) Current Protocols in Molecular BiologyAppendix 1C; DOI: 10.1002/0471142301.nsa01cs01, which is incorporated byreference in its entirety for all purposes), cell-penetrating peptides(e.g., Copolovici, Langel, Eriste, and Langel (2014) ACS Nano 2014 8(3), 1972-1994; DOI: 10.1021/nn4057269, which is incorporated byreference in its entirety for all purposes), electroporation (e.g Potter(2001) Current Protocols in Molecular Biology unit 10.15; DOI:10.1002/0471142735.im1015s03 and Kim et al (2014) Genome 1012-19.doi:10.1101/gr.171322.113, Kim et al. 2014 describe the AmazaNucleofector, an optimized electroporation system, both of thesereferences are incorporated by reference in their entirety for allpurposes), microinjection (e.g., McNeil (2001) Current Protocols in CellBiology unit 20.1; DOI: 10.1002/0471143030.cb2001s18, which isincorporated by reference in its entirety for all purposes), liposome orcell fusion (e.g., Hawley-Nelson and Ciccarone (2001) Current Protocolsin Neuroscience Appendix 1F; DOI: 10.1002/0471142301.nsa01fs10, which isincorporated by reference in its entirety for all purposes), mechanicalmanipulation (e.g. Sharon et al. (2013) PNAS 2013 110(6); DOI:10.1073/pnas.1218705110, which is incorporated by reference in itsentirety for all purposes) or other well-known technique for delivery ofnucleic acids to eukaryotic cells. Once introduced, the nucleic acid canbe transiently expressed episomally, or can be integrated into thegenome of the eukaryotic cell using well known techniques such asrecombination (e.g., Lisby and Rothstein (2015) Cold Spring HarbPerspect Biol. March 2; 7(3). pii: a016535. doi:10.1101/cshperspect.a016535, which is incorporated by reference in itsentirety for all purposes), or non-homologous integration (e.g., Deyleand Russell (2009) Curr Opin Mol Ther. 2009 August; 11(4):442-7, whichis incorporated by reference in its entirety for all purposes). Theefficiency of homologous and non-homologous recombination can befacilitated by genome editing technologies that introduce targeteddouble-stranded breaks (DSB). Examples of DSB-generating technologiesare CRISPR/Cas9, TALEN, Zinc-Finger Nuclease, or equivalent systems(e.g., Cong et al. Science 339.6121 (2013): 819-823, Li et al. Nucl.Acids Res (2011): gkr188, Gajet al. Trends in Biotechnology 31.7 (2013):397-405, all of which are incorporated by reference in their entiretyfor all purposes), transposons such as Sleeping Beauty (e.g., Singh etal (2014) Immunol Rev. 2014 January; 257(1):181-90. doi:10.1111/imr.12137, which is incorporated by reference in its entiretyfor all purposes), targeted recombination using, for example, FLPrecombinase (e.g., O'Gorman, Fox and Wahl Science (1991)15:251(4999):1351-1355, which is incorporated by reference in itsentirety for all purposes), CRE-LOX (e.g., Sauer and Henderson PNAS(1988): 85; 5166-5170), or equivalent systems, or other techniques knownin the art for integrating the nucleic acid into the eukaryotic cellgenome.

In an embodiment, the nucleic acid(s) encoding the Gα, Gβ, Gγ, adenylatecyclase, and the Olfactory Receptor are integrated into the eukaryotichost cell chromosome at a genomic safe harbor site, such as, forexample, the CCR5, AAVS1, human ROSA26, or PSIP1 loci for human cells.(Sadelain et al., Nature Rev. 12:51-58 (2012); Fadel et al., J. Virol.88(17):9704-9717 (2014); Ye et al., PNAS 111(26):9591-9596 (2014), allof which are incorporated by reference in their entirety for allpurposes.) Safe harbor sites for yeast cells, e.g., Saccharomycescerevisiae, include, for example, yeast Ty δ sequences. In anembodiment, the host cell is a human cell and the integration of thenucleic acid(s) encoding the Gα, Gβ, Gγ, adenylate cyclase, and theOlfactory Receptor at the CCR5 and/or PSIP1 locus is done using a geneediting system, such as, for example, CRISPR, TALEN, or Zinc-Fingernuclease systems. In an embodiment, the eukaryotic cell is aSaccharomyces cerevisiae cell and a CRISPR system is used to integratethe Gα, Gβ, Gγ, adenylate cyclase, and the Olfactory Receptor at Ty δlocus. In an embodiment, integration of the nucleic acid at safe harborloci using the CRISPR system also deletes a portion, or all, of the safeharbor loci. In an embodiment, Cas9 in the eukaryotic cell may bederived from a plasmid encoding Cas9, an exogenous mRNA encoding Cas9,or recombinant Cas9 polypeptide alone or in a ribonucleoprotein complex.(Kim et al (2014) Genome 1012-19. doi:10.1101/gr.171322.113; Wang et al(2013) Cell 153 (4). Elsevier Inc.: 910-18.doi:10.1016/j.cell.2013.04.025, both of which are incorporated byreference in their entirety for all purposes.)

Chemical means for introducing a polynucleotide into a host cell includecolloidal dispersion systems, such as macromolecule complexes,nanocapsules, microspheres, beads, and lipid-based systems includingoil-in-water emulsions, micelles, mixed micelles, and liposomes. Anexemplary colloidal system for use as a delivery vehicle in vitro and invivo is a liposome (e.g., an artificial membrane vesicle). Other methodsof state-of-the-art targeted delivery of nucleic acids are available,such as delivery of polynucleotides with targeted nanoparticles or othersuitable sub-micron sized delivery system.

Uses of Biosensors

In some embodiments, the nucleic acids encoding the components of thebiosensor (e.g., the Olfactory Receptor, G-proteins, and adenylatecyclase and/or other reporter) are placed in a suitable host cell (e.g.,Saccharomyces cerevisiae) and the host cells with the biosensor are usedfor detection of odorants. In some embodiments, the host cells are lysedand a membrane fraction is obtained that includes the OlfactoryReceptor, G-proteins, and adenylate cyclase (and/or other reporter). Inthis embodiment, the membrane fraction is used as the biosensor fordetection of odorants.

In some embodiments, the biosensors of the invention are used toidentify the Olfactory Receptors that interact with a mixture,composition, or molecule and the degree of interaction at each OlfactoryReceptor by the mixture, composition, or molecule. The identity ofOlfactory Receptors and the degree of interaction produces an aromagraphfor a mixture, composition, or molecule. In some embodiments, thebiosensors of the invention are used to deconstruct the aromagraph of amixture, composition, and/or molecule to identify which components orsubstituents of the mixture, composition, or molecule produce thearomagraph. In some embodiments, the aromagraph of a mixture isdeconstructed by removing components from the mixture and testing thecomponent minus mixture on the biosensor. Removed components thatcontribute to the aromagraph will be identified by changes in thearomagraph. Similarly, a composition can be deconstructed by removingcomponents and identifying changes to the aromagraph. In someembodiments, the removed component is also tested on the biosensors tocharacterize the aromagraph for the component. In some embodiments, thesubstituents on a molecule are changed in effort to identify whichsubstituents of the molecule interact with the Olfactory Receptor(s).

In some embodiments, aromagraphs are used to create a specification fora mixture, composition, and/or molecule. This aromagraph specificationcan be used for QC and QA of a product. In some embodiments, the productfor QC or QA includes, for example, a spice, seasoning, fragrance,perfume, food product, pet product. In some embodiments, aromagraphspecifications are used for the ingredients of a product. In thisembodiment, an ingredient can be exchanged for a different ingredient orfor the same ingredient from a different source as long as thearomagraph specification is met. In some embodiments, a new ingredientor the same ingredient from a different source will require slightmodification (e.g., addition of molecules) to meet the aromagraphspecification. The use of the biosensors of the invention andaromagraphs allows for rationale design and rationale substitution inthe creation and maintenance of products that rely upon aroma, scent,smell, odor, and/or taste. In some embodiments, the aromagraph for aproduct or composition is used to replace an ingredient in the productor composition that has become unavailable, expensive, or hard toacquire. In some embodiments, the aromagraph is used to replace anon-healthy ingredient with a healthy ingredient. In some embodiments,the aromagraph is used to replace a non-natural ingredient with anatural ingredient. In some embodiments, the aromagraph is used toreplace an ingredient with a less costly ingredient. In someembodiments, the aromagraph is used to replace a plurality ofingredients with fewer ingredients. In some embodiments, the product orcomposition is a flavor or fragrance, and the aromagraph of the flavoror fragrance is used to build a substitute recipe for the flavor orfragrance. The invention also relates to the new products made by thesenew formulations.

In some embodiments, the biosensors of the invention are used tostandardize scents, smells, aromas, odors, and/or taste. In someembodiments, aromagraphs are used to standardize scent, smell, aroma,odor, and/or a taste. In some embodiments, the biosensors of theinvention are used to quantify the interaction of a scent, smell, odor,aroma, and/or taste with the OR repertoire. These quantized interactionscan be used to describe a scent, smell, odor, aroma, and/or taste. Forexample, hundreds of vanilla flavoring or vanilla extract productsadvertise that they provide vanilla flavor (or smell, scent, aroma). Thebiosensors of the invention can quantify the interaction of thesevanilla products with the OR repertoire and permit standardization ofthese products on a functional basis. A core set of OR interactions willdefine the vanilla response, with many minor or side OR interactionsproducing the differences between these vanilla products.Standardization also allows the quantitative description of scent,smell, aroma, odor, and/or taste for purposes of branding, trademarks,and/or copyrights. Standardization also allows the quantitativedescription of new scents, smells, aromas, odors, and/or tastes. In someembodiments, the elements of flavor wheels are digitized intoAromagraphs and qualitative terms from the flavor wheel are associatedwith quantified interactions at Olfactory Receptors. See Google Imagesfor Flavor Wheels. For example, the flavor wheel descriptors caramel(honey, butterscotch, butter, soy sauce, chocolate, molasses), chemical(Sulphur dioxide, ethanol, acetic acid, ethyl acetate, wet wool, wetdog, Sulphur dioxide, burnt match, cabbage, skunk, garlic, mercaptan,hydrogen sulfide, rubbery, diesel, kerosene, plastic, tar), earthy(moldy, moldy cork, mushroom, dusty), floral (geranium, violet, rose,orange blossom), fruity (grapefruit, lemon, blackberry, raspberry,strawberry, black currant, cherry, apricot, peach, apple, pineapple,melon, banana, strawberry jam, raisin, prune, fig, methyl anthranilate),herbaceous or vegetative (cut green grass, bell pepper, eucalyptus,mint, green beans, asparagus, green olive, black olive, artichoke, hay,straw, tea, tobacco), microbiological (mousey, horsey, yoghurt, sweaty,sauerkraut, leesy, baker's yeast, nutty (walnut, hazelnut, almond),oxidized (sherry), pungent (menthol, alcohol), spic (licorice anise,black pepper, cloves), and woody (smokey, burnt toast, coffee,medicinal, phenolic, bacon, oak, cedar, vanilla) are quantified by theirOlfactory Receptor responses.

In some embodiments, the quantified scent, smell, odor, aroma, and/ortaste is used to identify the scent, smell, odor, aroma, and/or taste.In some embodiments, the quantified scent, smell, odor, aroma, and/ortaste is used to uniquely identify the scent, smell, odor, aroma, and/ortaste. In some embodiments, the quantified scent, smell, odor, aroma,and/or taste is used to communicate about the scent, smell, odor, aroma,and/or taste. In some embodiments, the quantified scent, smell, odor,aroma, and/or taste is used in a transaction for the scent, smell, odor,aroma, and/or taste. In some embodiments, quantified scent, smell, odor,aroma, and/or taste is used to describe a product or service. In someembodiments, quantified scent, smell, odor, aroma, and/or taste is usedto market a product or service. In some embodiments, quantified scent,smell, odor, aroma, and/or taste is used to sell a product or service.In some embodiments, quantified scent, smell, odor, aroma, and/or tasteis used to buy a product or service.

In some embodiments, an aromagraph is used to identify the scent, smell,odor, aroma, and/or taste. In some embodiments, an aromagraph is used touniquely identify the scent, smell, odor, aroma, and/or taste. In someembodiments, an aromagraph is used to communicate about the scent,smell, odor, aroma, and/or taste. In some embodiments, an aromagraph isused in a transaction for the scent, smell, odor, aroma, and/or taste.In some embodiments, an aromagraph is used to describe a product orservice. In some embodiments, an aromagraph is used to market a productor service. In some embodiments, an aromagraph is used to sell a productor service. In some embodiments, an aromagraph is used to buy a productor service.

In some embodiments, the biosensors of the invention are used toremediate a malodor. In some embodiments, the malodor arises in themanufacture of a product that has a characteristic scent, smell, aroma,odor, and/or taste. The lot of product with the malodor can be tested onthe biosensors of the invention and the aromagraph obtained compared tothe aromagraph specification for the product. This will identify new orchanged Olfactory Receptor interactions causing the malodor. In someembodiments, this malodor is remediated by deconstructing the productwith the malodor to identify which component is causing the malodor. Insome embodiments, the malodor is caused by new interactions at newOlfactory Receptors. In this embodiment, the malodor could be remediatedby adding an inhibitor(s) that blocks the new Olfactory Receptor(s) frombeing activated. Alternatively, a molecule could be added that activatesother Olfactory Receptors which combined with the new Olfactory Receptorstimulation turns the perception of the malodor into a pleasant, minor,or not perceived smell, scent, aroma, odor, and/or taste. In someembodiments, the malodor is caused by an imbalance in components thatchanges the interactions at the Olfactory Receptors in the aromagraphspecification. In this embodiment, the malodor may be removed bybalancing the components of the product to restore the aromagraphspecification of the product. In some embodiments, the aromagraph may bebalanced by increasing the amount of certain ingredients to rationallybalance the aromagraph to the product specification based on thearomagraphs of the ingredients. In some embodiments, the aromagraph maybe balanced by adding molecules known to stimulate or inhibit certainOlfactory Receptors so as to bring the aromagraph of the product withthe malodor back into balance with its aromagraph specification.

In some embodiments, the malodor is associated with sports equipment,clothing, or shoes. The malodor can be characterized on the biosensorsof the invention to create a malodor aromagraph. In some embodiments,the malodor is remediated by adding an inhibitor(s) that blocksOlfactory Receptor(s) in the malodor aromagraph from being activated.Alternatively, a molecule could be added that activates other OlfactoryReceptors which combined with the Olfactory Receptors stimulated by themalodor turns the perception of the malodor into a pleasant, minor, ornot perceived smell, scent, aroma, odor, and/or taste.

In some embodiments, the biosensors of the invention are used to make amixture, composition, or molecule with a desired aromagraphspecification. For example, the biosensors of the invention can be usedto deconstruct the aromagraph for cadaverine and/or putrescine. Thesecompounds are known to cause avoidance behavior in some subjects.Deconstructing the contribution of certain moieties to the cadaverineand putrescine aromagraphs may identify the Olfactory Receptorsresponsible for the avoidance behavior of these molecules. Rationaledesign can then be used to make a mixture, composition, or molecule thatcan stimulate these Olfactory Receptors to cause the avoidance behaviorwithout the foul smell of cadaverine or putrescine. Such a rationallydesigned aroma, scent, smell or odor could be used in an aerosol tocause avoidance behavior and suppress appetite in subjects.Alternatively, the rationally designed aroma, scent, smell or odor couldbe used to keep subjects away from an area as the avoidance behaviorcaused by these Olfactory Receptors would cause subjects to avoid thescent, smell, aroma, and/or odor.

In some embodiments, the rationally designed aroma, scent, smell, odor,and/or taste is reverse engineered from a known aroma, scent, smell,odor, and/or taste. This reverse engineering can produce a newformulation for making the known aroma, scent, smell, odor, and/ortaste. In some embodiments, the rationally designed aroma, scent, smell,odor, and/or taste includes, for example, new car smell, perfumes,fragrances, or flavors. In some embodiments, the perfume or fragrance isarchived in the Osmothèque. In some embodiments, an aromagraph for anarchived fragrance or perfume from the Osmothèque is used to create newformulation and recipe for making the fragrance or perfume. In someembodiments, the recipe for the fragrance or perfume from the Osmothèquehas been lost, and the biosensors and aromagraphs of the invention areused to create a recipe and formulation for making the fragrance orperfume.

In some embodiments, the rationally designed aroma, scent, smell and/orodor is delivered to an environment by placing it in a material(plastic, leather, cloth, wood, wax, etc.) from which the aroma, scent,smell and/or odor is slowly released over time. In some embodiments, thearoma, scent, smell and/or odor is released from the material when it issubjected to a different set of conditions. For example, the aroma,scent, smell and/or odor could be placed in a material where it isreleased upon heating of the material (e.g., similar to glad plug-ins).Other changes in conditions besides temperature can also be used torelease the aroma, scent, smell and/or odor, including, for example, asolvent, pH, airflow, light, etc. can be used to release the aroma,scent, smell and/or odor from the material.

In some embodiments, aromagraph specifications are designed to accountfor differences in socioeconomic, cultural, geographic, or the palatesof other groupings of certain subjects. In some embodiments, aromagraphspecifications are created for aromascapes or odorscapes that produce adesired response or state in a subject from certain socioeconomic,cultural, geographic, and/or other groupings. In some embodiments, thearomascape specification is designed to relax, attract, repel, etc.subjects from certain socioeconomic, cultural, geographic, and/or othergroups. In some embodiments, aromagraph specification are created forproducts so subjects in certain socioeconomic, cultural, geographic,and/or other groupings will have a desired response to the product. Insome embodiments, aromagraph specifications are created or defined aspart of product or service branding.

In some embodiments, the biosensors and aromagraphs of the invention areused to tailor a product for the palates of consumers in differentgeographic locations. In some embodiments, a products aroma, scent,smell, odor, and/or taste can be designed to be appealing to consumersin different geographic locations. In some embodiments, aromagraphs fora class or group of products from different cultures and/or geographiclocations can be used to tailor new products of the class or group fordifferent geographic/cultural markets. For example, a chocolate productcan be designed for Middle Eastern or Indian markets by usingaromagraphs for similar types of products from these geographic areas toidentify OR interactions that are favored by consumers for those typesof products. Using this information on OR receptors and aromagraphs, achocolate product can be rationally designed for these differentmarkets.

In some embodiments, the biosensors of the invention are used to detectand diagnose disease. Many diseases are associated with odors or smellsthat can be used to diagnose the disease. For example, certain lung,liver, kidney and digestive diseases can be detected from a patient'sbreath, diabetes, schizophrenia, Parkinson's, and certain infectiousdiseases (tuberculosis and typhoid) can be detected by a patient's odor,and some cancers can be detected by the olfactory repertoire of canines.In some embodiments, odors, scent, and/or smell associated with apatient's skin, sweat, hair, saliva, and other body secretions (e.g.,ear wax) can be associated with disease diagnosis. In some embodiments,a biosensor of the invention is used to create aromagraphs of patient'swith diseases that can be detected by odor, scent, and/or smell. In someembodiments, the aromagraph is based on a human repertoire of OlfactoryReceptors. In some embodiments, the aromagraph is based on a caninerepertoire of Olfactory Receptors. In some embodiments, the aromagraphis based on a mouse or rat repertoire of Olfactory Receptors. In someembodiments, the aromagraph is based on a mammalian repertoire ofOlfactory Receptors. Patients can then be diagnosed for disease bytaking odor, breath or other samples and screening them to see whetherthe aromagraph for a certain disease is detected.

In some embodiments, the biosensors of the invention are used toidentify sets of OR that are associated with disease. In someembodiments, panels of ligands for these disease specific OR(s) are madeand can be used to monitor a disease by the changes in a patient'sresponse at the OR associated with the disease. For example, a poorsense of smell is one of the early warning signs of Alzheimer's. Thedegradation of the sense of smell is associated with both a loss of theability of the brain to sense some OR and the loss of Olfactory Receptormemory (association of a smell with the stimulation of certain OR). Theloss of OR response and OR memory can be used as an early warning signfor Alzheimer's, and can also be used to monitor response toanti-Alzheimer's treatment, as the loss of smell is reversible in somecases. In some embodiments, patients at risk for Alzheimer's can betested for loss of smell at disease associated ORs, and for OR memory.In some embodiments, patients who reach a certain age can be screenedfor loss of smell at disease associated ORs and for OR memory. In someembodiments, panels of odorants can be used to monitor a patient's senseof smell at the disease associated ORs. In some embodiments, the panelof odorants have different interactions at the disease associated ORfrom strong to weak interactions. In some embodiments, the biosensor ofthe invention can be used to identify disease associated ORs and toidentify ligands that can be used to diagnose early Alzheimer's.

In some embodiments, the biosensors of the invention are used in drugdiscovery. In some embodiments, the biosensors of the invention are usedto design the taste, smell, odor, scent, and/or aroma of a drug and/orpharmaceutical composition. In some embodiments, the biosensors of theinvention are used to identify and mask a taste, smell, odor, scent,and/or aroma associated with a drug and/or pharmaceutical composition.In some embodiments, the taste, smell, odor, scent, and/or aroma whichis masked produces a negative response in certain subjects. In someembodiments, the taste, smell, odor, scent, and/or aroma which is maskedproduces a positive or addictive response in certain subjects. In someembodiments, the biosensors of the invention are used to designabuse-deterrent formulations. In some embodiments, the adversantformulations are used for opioid drugs including, for example,hydrocodone (Vicodin), oxycodone (OxyContin, Percocet, Roxicodone,Oxecta), morphine (Kadian, Avinza), codeine, buprenorphine (Buprenex,Butrans), butorphanol (Stadol), hydromorphone (Dilaudid, Hydrostat,Exalgo), levorphanol (Levo-Dromoran), meperidine (Demerol), methadone(Dolophine, Methadose), nalbuphine (Nubain), oxymorphone (Numorphan),pentazocine (Talwin), propoxyphene (Cotanal-65, Darvon), fentanyl(Sublimaze, Actiq, Durogesic, Fentora, Matrifen, Hadid, Onsolis,Instanyl, Abstral, Lazanda), tramadol (Ultram), and tapentadol(Nucynta). In some embodiments, an adversant is included in theformulation that produces a taste, smell, odor, scent, and/or aroma thatproduces an avoidance behavior or other strongly negative reaction bysubjects. In some embodiments, the adversant is comprised of two or morecomponents that when sensed together produce the negative reaction, butwhen sensed individually do not induce the negative reaction. In someembodiments, the two components can be engineered in the abuse deterrentformulation to be released at different times, but when the formulationis crushed or extracted to abuse the drug, this releases both componentsto form the adversant. In some embodiments, the adversant formed whenthe two components combine becomes a gas at room temperature, or becomesa gas after the components mix and the drug formulation is heated.

In some embodiments, an adversant is designed for application toproducts. In some embodiments, the adversant is applied to products,including, for example, tobacco or marijuana. In these embodiments, theadversant would be formulated to adsorb into the plant tissues so it isreleased along when the tobacco or marijuana is used, for example, theadversant could be designed to be released when the tobacco or marijuanais burned. In some embodiments, the adversant is one that alters theinteraction of the product with the OR of the subject resulting in anadverse aroma, scent, smell, odor, and/or taste.

In some embodiments, the biosensors of the invention are used toidentify panels of odorants for groups of ORs. In some embodiments, apanel of odorants is used to monitor an individual's sense of smelland/or taste. In some embodiments, a panel of odorants is used tomonitor the sense of smell and/or taste in a group of individuals, e.g.,a panel of individuals for testing smells and/or tastes of products. Insome embodiments, the panel of odorants is used to set a baseline for anindividual. In some embodiments, the panel of odorants is used to set aspecification for a tasting/smelling panel of individuals. In someembodiments, the specification is used to set a panel of individuals foreach tasting/smelling panel study.

In some embodiments, the biosensor of the invention is used to identifythe components of a taste, smell, odor, scent, and/or aroma so thatcomponents of various tastes, smells, odors, scents, and/or aromas arereleased together or sequentially to produce desired effects at desiredtimes. In some embodiments, a series of components are released so thatat a first time period, the components produce a taste, smell, odor,scent, and/or aroma that produces desired response in the first timeperiod, and then another or different group of components is released ata second time period to change or introduce a new taste, smell, odor,scent, and/or aroma to produce a different desired response in thesecond time period. This changing of a taste, smell, odor, scent, and/oraroma in different time periods can be repeated for as many time periodsas desired.

In some embodiments, the invention relates to panels or libraries ofodorants with known Aromagraphs generated from the biosensors of theinvention. These panels or libraries of odorants can be mixed andmatched to make a composition that will have a desired Aromagraph. Insome embodiments, a device contains the panel or library of odorants andcan make compositions that will meet a specific Aromagraph by mixingodorants from the panel or library. In some embodiments, the devicegenerates an Aromagraph from a composition, and makes a composition thatmatches the Aromagraph of the tested composition. In some embodiments,the device makes the composition matching the Aromagraph of the testedcomposition at a site remote from where the testing of the compositionoccurs. In some embodiments, the device makes the composition matchingthe Aromagraph of the tested composition at a site remote from where thedesired Aromagraph is inputted.

All publications and patents cited in this specification are hereinincorporated by reference as if each individual publication or patentwere specifically and individually indicated to be incorporated byreference and are incorporated herein by reference to disclose anddescribe the methods and/or materials in connection with which thepublications are cited.

EXAMPLES Example 1: Making a Biosensor in a Yeast Cell

The N-terminal 55 amino acids of the human OR6A2 receptor are fused tothe N-terminal region of the human OR1A1 receptor in place of theN-terminal 55 amino acids of OR1A1 to give a biosensor OlfactoryReceptor with the sequence:

(SEQ ID NO: 12) MEWRNHSGRV SEFVLLGFPA PAPLQVLLFA LLLLAYVLVLTENTLIIMAI RNHSTHNPMY FLLANLSLVD IFFSSVTIPKMLANHLLGSK SISFGGCLTQ MYFMIALGNT DSYILAAMAYDRAVAISRPL HYTTIMSPRS CIWLIAGSWV IGNANALPHTLLTASLSFCG NQEVANFYCD ITPLLKLSCS DIHFHVKMMY LGVGIFSVPL LCIIVSYIRV FSTVFQVPST KGVLKAFSTCGSHLTVVSLY YGTVMGTYFR PLTNYSLKDA VITVMYTAVTPMLNPFIYSL RNRDMKAALR KLFNKRISS 

A nucleic acid encoding this Olfactory Receptor is engineered into ayeast cell that has been previously engineered to express the human Gprotein subunits Gα, Gβ, and Gγ, and adenylate cyclase. Yeast wereengineered with constructs that placed the human Gα, Gβ, and Gγsubunits:

(SEQ ID NO: 13) MGCLGGNSKTTEDQGVDEKERREANKKIEKQLQKERLAYKATHRLLLLGAGESGKSTIVKQMRILHVNGFNPEEKKQKILDIRKNVKDAIVTIVSAMSTIIPPVPLANPENQFRSDYIKSIAPITDFEYSQEFFDHVKKLWDDEGVKACFERSNEYQLIDCAQYFLERIDSVSLVDYTPTDQDLLRCRVLTSGIFETRFQVDKVNFHMFDVGGQRDERRKWIQCFNDVTAIIYVAACSSYNMVIREDNNTNRLRESLDLFESIWNNRWLRTISIILFLNKQDMLAEKVLAGKSKIEDYFPEYANYTVPEDATPDAGEDPKVTRAKFFIRDLFLRISTATGDGKHYCYPHFTCAVDTENIRRVFNDCRDIIQRMHLKQYELL (SEQ ID NO: 14)MSELEQLRQEAEQLRNQIRDARKACGDSTLTQITAGLDPVGRIQMRTRRTLRGHLAKIYAMHWGTDSRLLVSASQDGKLIIWDSYTTNKVHAIPLRSSWVMTCAYAPSGNFVACGGLDNICSIYSLKTREGNVRVSRELPGHTGYLSCCRFLDDNQIITSSGDTTCALWDIETGQQTVGFAGHSGDVMSLSLAPDGRTFVSGACDASIKLWDVRDSMCRQTFIGHESDINAVAFFPNGYAFTTGSDDATCRLFDLRADQELLMYSHDNIICGITSVAFSRSGRLLLAGYDDFNCNIWDAMKGDRAGVLAGHDNRVSCLGVTDDGMAVATGSWDSFLKIWN (SEQ ID NO: 15)MSGSSSVAAMKKVVQQLRLEAGLNRVKVSQAAADLKQFCLQNAQHDPLLT GVSSSTNPFRPQKVCSFLunder the control of either the yeast GALI or GAL10 promoter. The yeaststrain was also engineered to express human adenylate cyclase (humanadenylate cyclase type 5, NP 001186571.1):

(SEQ ID NO: 16) MPRNQGFSEPEYSAEYSAEYSVSLPSDPDRGVGRTHEISVRNSGSCLCLPRFMRLTFVPESLENLYQTYFKRQRHETLLVLVVFAALFDCYVVVMCAVVFSSDKLASLAVAGIGLVLDIILFVLCKKGLLPDRVTRRVLPYVLWLLITAQIFSYLGLNFARAHAASDTVGWQVFFVFSFFITLPLSLSPIVIISVVSCVVHTLVLGVTVAQQQQEELKGMQLLREILANVFLYLCAIAVGIMSYYMADRKHRKAFLEARQSLEVKMNLEEQSQQQENLMLSILPKHVADEMLKDMKKDESQKDQQQFNTMYMYRHENVSILFADIVGFTQLSSACSAQELVKLLNELFARFDKLAAKYHQLRIKILGDCYYCICGLPDYREDHAVCSILMGLAMVEAISYVREKTKTGVDMRVGVHTGTVLGGVLGQKRWQYDVWSTDVTVANKMEAGGIPGRVHISQSTMDCLKGEFDVEPGDGGSRCDYLEEKGIETYLIIASKPEVKKTATQNGLNGSALPNGAPASSKSSSPALIETKEPNGSAHSSGSTSEKPEEQDAQADNPSFPNPRRRLRLQDLADRVVDASEDEHELNQLLNEALLERESAQVVKKRNTFLLSMRFMDPEMETRYSVEKEKQSGAAFSCSCVVLLCTALVEILIDPWLMTNYVTFMVGEILLLILTICSLAAIFPRAFPKKLVAFSTWIDRTRWARNTWAMLAIFILVMANVVDMLSCLQYYTGPSNATAGMETEGSCLENPKYYNYVAVLSLIATIMLVQVSHMVKLTLMLLVAGAVATINLYAWRPVFDEYDHKRFREHDLPMVALEQMQGFNPGLNGTDRLPLVPSKYSMTVMVFLMMLSFYYFSRHVEKLARTLFLWKIEVHDQKERVYEMRRWNEALVTNMLPEHVARHFLGSKKRDEELYSQTYDEIGVMFASLPNFADFYTEESINNGGIECLRFLNEIISDFDSLLDNPKFRVITKIKTIGSTYMAASGVTPDVNTNGFASSNKEDKSERERWQHLADLADFALAMKDTLTNINNQSENNFMLRIGMNKGGVLAGVIGARKPHYDIWGNTVNVASRMESTGVMGNIQVVEETQVILREYGFRFVRRGPIFVKGKGELLTFFLKGRDKLATFPNGPSVTLPHQVVDNS Or human adenylate cyclase 3 (UniProtKB: 060266):

(SEQ ID NO: 17) MPRNQGFSEPEYSAEYSAEYSVSLPSDPDRGVGRTHEISVRNSGSCLCLPRFMRLTFVPESLENLYQTYFKRQRHETLLVLVVFAALFDCYVVVIVICAVVFSSDKLASLAVAGIGLVLDIILFVLCKKGLLPDRVTRRVLPYVLWLLITAQIFSYLGLNFARAHAASDTVGWQVFFVFSFFITLPLSLSPIVIISVVSCVVHTLVLGVTVAQQQQEELKGMQLLREILANVFLYLCAIAVGIMSYYMADRKHRKAFLEARQSLEVKMNLEEQSQQQENLMLSILPKHVADEMLKDMKKDESQKDQQQFNTMYMYRHENVSILFADIVGFTQLSSACSAQELVKLLNELFARFDKLAAKYHQLRIKILGDCYYCICGLPDYREDHAVCSILMGLAMVEAISYVREKTKTGVDMRVGVHTGTVLGGVLGQKRWQYDVWSTDVTVANKMEAGGIPGRVHISQSTMDCLKGEFDVEPGDGGSRCDYLEEKGIETYLIIASKPEVKKTATQNGLNGSALPNGAPASSKSSSPALIETKEPNGSAHSSGSTSEKPEEQDAQADNPSFPNPRRRLRLQDLADRVVDASEDEHELNQLLNEALLERESAQVVKKRNTFLLSMRFMDPEMETRYSVEKEKQSGAAFSCSCVVLLCTALVEILIDPWLMTNYVTFMVGEILLLILTICSLAAIFPRAFPKKLVAFSTWIDRTRWARNTWAMLAIFILVMANVVDMLSCLQYYTGPSNATAGMETEGSCLENPKYYNYVAVLSLIATIMLVQVSHMVKLTLMLLVAGAVATINLYAWRPVFDEYDHKRFREHDLPMVALEQMQGFNPGLNGTDRLPLVPSKYSMTVMVFLMMLSFYYFSRHVEKLARTLFLWKIEVHDQKERVYEMRRWNEALVTNMLPEHVARHFLGSKKRDEELYSQTYDEIGVMFASLPNFADFYTEESINNGGIECLRFLNEIISDFDSLLDNPKFRVITKIKTIGSTYMAASGVTPDVNTNGFASSNKEDKSERERWQHLADLADFALAMKDTLTNINNQSFNNFMLRIGMNKGGVLAGVIGARKPHYDIWGNTVNVASRMESTGVNIGNIQVVEETQVILREYGFRFVRRGPIFVKGKGELLTFFLKGRDKLATFPNGPSVTLPHQVVDNS under the control of either the yeast GAL I or GAL10 promoters.

Biosensors with yeast cells expressing the hybrid olfactory receptor,the human Gα, Gβ, and Gγ subunits, and human adenylate cyclase aretested for expression of the components and for signal transduction bythe hybrid OR.

Example 2: Using Real Time Detection to Quantitate Ligand Binding at aRepertoire of Olfactory Receptors

A plurality of biosensors as described in Example 1, are used for humanOlfactory Receptors from OR Family OR7. The Yeast cells with the OR7family Olfactory Receptors are also genetically modified to include arecombinant GFP gene expressed by a control region activated by cAMP.Thus, when the biosensor is activated by an odorant, the biosensor willproduce GFP and activity can be monitored by fluorescence.

Olfactory Receptors in the OR7 family are receptors for mammalianpheromones such as those related to androstenone. A panel of odorants isscreened against a panel of androstenone related molecules, including,androstadienol (5,16-androstadien-3β-ol), androstadienone(androsta-4,16-dien-3-one), androstanol (5α-androst-16-en-3α-ol), andestratetraenol (estra-1,3,5(10),16-tetraen-3-ol).

Yeast cells expressing different members of the OR7 family of OlfactoryReceptors are placed into separate wells or containers, interrogatedwith individual odorants from the panel, and fluorescence readings aremade at time points 0, 10 seconds, 20 seconds, 30 seconds, 1 minute, 2minutes, 3 minutes, 4 minutes, 5 minutes, 10 minutes, 20 minutes, 30minutes, 40 minutes, 50 minutes, and 1 hour.

Example 3: Making a Biosensor with Human Olfactory Receptor OR1A1

The full length human olfactory receptor OR1A1 was used to make theexpression plasmids NIXp218 and NIXp354. In both NIXp218 and NIXp354,the nucleic acid encoding OR1A1 is under the control of a GAL1-10promoter. A general plasmid map for the OR constructs is shown inFIG. 1. In NIXp354, OR1A1 is fused at its N-terminal end with a FLAG tag(SEQ ID NO:1), and at its C-terminal end with the coding sequence forred fluorescent protein (RFP).

Plasmid NIXp354 or NIXp218 was placed in a haploid Saccharomycescerevisiae (MATa strain). Expression of OR1A1 from NIXp354 is monitoredusing the FLAG tag to measure expression (using an immunoassay) andcellular localization of the OR1A1 is monitored by fluorescence from theRFP. Function of the biosensor is assessed by mating the Saccharomycescerevisiae strain with NIXp218 to the complementary Saccharomycescerevisiae strain (MATα) which is modified with the human G proteinsubunits Gα, Gβ, and Gγ, and adenylate cyclase. Mating together thesetwo yeast strains brings the OR1A1 receptor into functional associationwith human G protein subunits Gα, Gβ, and Gγ, and adenylate cyclase.Function of the OR1A1 receptor can be assessed by a cAMP assay followingstimulation of the OR1A1 receptor.

Example 4: Making a Biosensor with Human Olfactory Receptor OR2J2

The full length human olfactory receptor OR2J2 was used to make theexpression plasmids NIXp219 and NIXp352. In both NIXp219 and NIXp352,the nucleic acid encoding OR2J2 is under the control of a GAL1-10promoter. A plasmid map for the OR constructs is shown in FIG. 1. InNIXp352, OR2J2 is fused at its N-terminal end with a FLAG tag (SEQ IDNO:1), and at its C-terminal end with the coding sequence for redfluorescent protein (RFP).

Plasmid NIXp352 or NIXp219 was placed in haploid Saccharomycescerevisiae (MATa strain). Expression of OR2J2 from NIXp352 is monitoredusing the FLAG tag to measure expression (using an immunoassay) andcellular localization of the OR2J2 is monitored by fluorescence from theRFP. Function of the biosensor is assessed by mating the Saccharomycescerevisiae strain with NIXp219 to the complementary Saccharomycescerevisiae strain (MATα) which is modified with the human G proteinsubunits Gα, Gβ, and Gγ, and adenylate cyclase. Mating together thesetwo yeast strains brings the OR2J2 receptor into functional associationwith human G protein subunits Gα, Gβ, and Gγ, and adenylate cyclase.Function of the OR2J2 receptor can be assessed by a cAMP assay followingstimulation of the OR2J2 receptor.

Example 5: Making a Biosensor with Human Olfactory Receptor OR2W1

The full length human olfactory receptor OR2W1 was used to makeexpression plasmids NIXp220 and NIXp351. In both NIXp220 and NIXp351,the nucleic acid encoding OR2W1 is under the control of a GAL1-10promoter. A plasmid map for the OR constructs is shown in FIG. 1. InNIXp351, OR2W1 is fused at its N-terminal end with a FLAG tag (SEQ IDNO:1), and at its C-terminal end with the coding sequence for redfluorescent protein (RFP).

Plasmid NIXp351 or NIXp220 was placed in haploid Saccharomycescerevisiae (MATa strain). Expression of OR2W1 from NIXp351 is monitoredusing the FLAG tag to measure expression (using an immunoassay) andcellular localization of the OR2W1 is monitored by fluorescence from theRFP. Function of the biosensor is assessed by mating the Saccharomycescerevisiae strain with NIXp220 to the complementary Saccharomycescerevisiae strain (MATα) which is modified with the human G proteinsubunits Gα, Gβ, and Gγ, and adenylate cyclase. Mating together thesetwo yeast strains brings the OR2W1 receptor into functional associationwith human G protein subunits Gα, Gβ, and Gγ, and adenylate cyclase.Function of the OR2W1 receptor can be assessed by a cAMP assay followingstimulation of the OR2W1 receptor.

Example 6: Making a Biosensor with Human Olfactory Receptor OR5P3

The full length human olfactory receptor OR5P3 was used to make theexpression plasmids NIXp217 and NIXp353. In both NIXp217 and NIXp353,the nucleic acid encoding OR5P3 is under the control of a GAL1-10promoter. A plasmid map for the OR constructs is shown in FIG. 1. InNIXp353, OR5P3 is fused at its N-terminal end with a FLAG tag (SEQ IDNO:1), and at its C-terminal end with the coding sequence for redfluorescent protein (RFP).

Plasmid NIXp353 or NIXp217 was placed in haploid Saccharomycescerevisiae (MATa strain). Expression of OR5P3 from NIXp353 is monitoredusing the FLAG tag to measure expression (using an immunoassay) andcellular localization of the OR5P3 is monitored by fluorescence from theRFP. Function of the biosensor is assessed by mating the Saccharomycescerevisiae strain with NIXp217 to complementary Saccharomyces cerevisiaestrain (MATα) which is modified with the human G protein subunits Gα,Gβ, and Gγ, and adenylate cyclase. Mating together these two yeaststrains brings the OR5P3 receptor into functional association with humanG protein subunits Gα, Gβ, and Gγ, and adenylate cyclase. Function ofthe OR5P3 receptor can be assessed by a cAMP assay following stimulationof the OR5P3 receptor.

Example 7: Making a Biosensor with Human Olfactory Receptor OR6A2

The full length human olfactory receptor OR6A2 was used to make theexpression plasmid NIXp239. In NIXp239 the nucleic acid encoding OR6A2is under the control of a GAL1-10 promoter. A plasmid map for the ORconstruct is shown in FIG. 1.

Plasmid NIXp239 was placed in haploid Saccharomyces cerevisiae (MATastrain). Function of the biosensor is assessed by mating theSaccharomyces cerevisiae strain with NIXp239 to the complementarySaccharomyces cerevisiae strain (MATα) which is modified with the humanG protein subunits Gα, Gβ, and Gγ, and adenylate cyclase. Matingtogether these two yeast strains brings the OR6A2 receptor intofunctional association with human G protein subunits Gα, Gβ, and Gγ, andadenylate cyclase. Function of the OR6A2 receptor can be assessed by acAMP assay following stimulation of the OR6A2 receptor.

Example 8: Making a Cell Extract with a Biosensor

In this example the yeast cells with biosensors made from OR1A1, OR2J2,OR2W1, OR5P3, and OR6A2 from Examples 3-7 are used. Yeast cells with theOlfactory Receptor, G-proteins and adenylate cyclase are lysed withglass beads in a blender. Cell debris is removed by centrifuging thelysate at 600×g. The remaining lysate is centrifuged in anultracentrifuge (104,300×g) to obtain the membrane fraction with theOlfactory Receptor, G-proteins and adenylate cyclase. The membranefraction is resuspended and placed into a multiwell plate for detectionof odorants.

All publications and patents cited in this specification are hereinincorporated by reference as if each individual publication or patentwere specifically and individually indicated to be incorporated byreference and are incorporated herein by reference to disclose anddescribe the methods and/or materials in connection with which thepublications are cited.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

We claim:
 1. A method of making an Aromagraph of a product including an odorant, comprising the steps of: obtaining a plurality of biosensors, wherein each biosensor comprises a mammalian Olfactory Receptor, a G protein subunit Gα, a G protein subunit Gβ, and a G protein subunit Gγ, and a reporter, wherein binding of the odorant to the mammalian Olfactory Receptor sends a signal through the G protein subunits resulting in a signal from the reporter, wherein at least some of the biosensors have a different mammalian Olfactory Receptor; introducing the product to the plurality of biosensors; measuring the signal from the reporter for each of the plurality of biosensors; and generating a digital representation of the reporter signal for each of the plurality of biosensors to thereby make the Aromagraph.
 2. The method of claim 1, wherein the product is made of a plurality of components, and the Aromagraph is made for each component of the product.
 3. The method of claim 2, wherein the Aromagraph is made for a combination of the components of the product.
 4. A method of describing a product including one or more odorants, wherein the method comprises quantifying an interaction of the one or more odorants with a plurality of olfactory receptors, and wherein quantifying is accomplished using the Aromagraph made by the method of claim
 1. 5. The method of claim 1, wherein the biosensor is in a host cell selected from the group consisting of an Aspergillus, a Trichoderma, a Saccharomyces, a Chrysosporium, a Klyuveromyces, a Candida, a Pichia, a Debaromyces, a Hansenula, a Yarrowia, a Zygosaccharomyces, a Schizosaccharomyces, a Penicillium, and a Rhizopus.
 6. The method of claim 5, wherein the biosensor is in a membrane fraction of a host cell.
 7. The method of claim 1, wherein the biosensor further comprises an adenylate cyclase and wherein a nucleic acid encoding the reporter is operably linked to a control region responsive to cAMP.
 8. The method of claim 7, wherein the reporter is an optical reporter.
 9. The method of claim 8, wherein the optical reporter is a fluorescent protein.
 10. The method of claim 9, wherein the fluorescent reporter is a green fluorescent protein, a blue fluorescent protein, a yellow fluorescent protein, or a cyan fluorescent protein.
 11. The method of claim 8, wherein the optical reporter is an enzyme or a luminescent protein.
 12. The method of claim 1, wherein the biosensor comprises a membrane fraction of a host cell.
 13. The method of claim 1, wherein the product is in the gaseous phase.
 14. The method of claim 4, wherein the product is made of a plurality of components, and the Aromagraph is made for each component of the product.
 15. The method of claim 4, wherein the product is made of a plurality of components, and the Aromagraph is made for a combination of the components of the product. 